Transgenic GPCR expressing animals

ABSTRACT

The invention provides isolated nucleic acids molecules, designated muscarinic acetylcholine receptor 6 (“mACHR-6”) nucleic acid molecules, which encode polypeptides involved in the modulation of acetylcholine responses in acetylcholine responsive cells. The invention also provides antisense nucleic acid molecules, expression vectors containing mACHR-6 nucleic acid molecules, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which an mACHR-6 gene has been introduced or disrupted. The invention still further provides isolated mACHR-6 polypeptides, fusion polypeptides, antigenic peptides, and anti-mACHR-6 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.Ser. No. 08/985,090, filed Dec. 4, 1997, now pending. The contents ofthis co-pending patent application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Muscarinic receptors, so named because the actions ofacetylcholine on such receptors are similar to those produced by themushroom alkaloid muscarine, mediate most of the inhibitory andexcitatory effects of the neurotransmitter acetylcholine in the heart,smooth muscle, glands and in neurons (both presynaptic and postsynaptic)in the autonomic and the central nervous system (Eglen, R. and Watson,N. (1996) Pharmacology & Toxicology 78:59-68). The muscarinic receptorsbelong to the G protein-coupled receptor superfamily (Wess, J. et al.(1990) Comprehensive Medicinal Chemistry 3:423-491). Like all other Gprotein-coupled receptors, the muscarinic receptors are predicted toconform to a generic protein fold consisting of seven hydrophobictransmembrane helices joined by alternative intracellular andextracellular loops, an extracellular amino-terminal domain, and acytoplasmic carboxyl-terminal domain. The mammalian muscarinic receptorsdisplay a high degree of sequence identity, particularly in thetransmembrane domains, sharing approximately 145 invariant amino acids(Wess, J. (1993) TIPS 14:308-313). Moreover, all of the mammalianmuscarinic receptors contain a very large third cytoplasmic loop which,except for the membrane-proximal portions, displays virtually nosequence identity among the different family members (Bonner, T. I.(1989) Trends Neurosci. 12:148-151). Ligand binding to the receptor isbelieved to trigger conformational changes within the helical bundle,which are then transmitted to the cytoplasmic domain, where theinteraction with specific G proteins occurs.

[0003] Molecular cloning studies have revealed the existence of fivemolecularly distinct mammalian muscarinic receptor proteins, termed theM₁-M₅ receptors (Bonner, T. I. (1989) Trends Neurosci. 12:148-151; andHulme, E. C. et al. (1990) Annu. Rev. Pharmacol. Toxicol. 30:633-673).The M₁ receptor is expressed primarily in the brain (cerebral cortex,olfactory bulb, olfactory tubercle, basal forebrain/septum, amygdala,and hippocampus) and in exocrine glands (Buckley, N. J. et al. (1988) J.Neurosci. 8:4646-4652). The M₂ receptor is expressed in the brain(olfactory bulb, basal forebrain/septum, thalamus and amygdala), and inthe ileum and the heart. The M₃ receptor is expressed in the brain(cerebral cortex, olfactory tubercle, thalamus and hippocampus) thelung, the ileum, and in exocrine glands. The M₄ receptor is expressed inthe brain (olfactory bulb, olfactory tubercle, hippocampus and striatum)and in the lung. Finally, the M₅ receptor is expressed primarily in thebrain (substantia nigra) (Hulme, E. C. et al. (1990) A. Rev. Pharmac.Toxic. 30:633-673).

[0004] The two enzymes with which muscarinic receptors interact mostdirectly are adenylate cyclase and phospholipase C. Studies with clonedreceptors have shown that the M₁, M₃, and M₅ muscarinic receptors arecoupled to the types of G proteins known as Go (a stimulatory proteinlinked to phospholipase C) or Gq and that their activation results inthe activation of phospholipase C. The M₂ and M₄ muscarinic receptorsare coupled to a Gi protein (an inhibitory protein linked to adenylatecyclase), and their activation results in the inhibition of adenylatecyclase. Through these signal transduction pathways, the muscarinicreceptors are responsible for a variety of physiological functionsincluding the regulation of neurotransmitter release (includingacetylcholine release) from the brain, the regulation of digestiveenzyme and insulin secretion in the pancreas, the regulation of amylasesecretion by the parotid gland, and the regulation of contraction incardiac and smooth muscle (Caulfield, M. P. (1993) Pharmac. Ther.58:319-379).

SUMMARY OF THE INVENTION

[0005] This invention provides a novel nucleic acid molecule whichencodes a polypeptide, referred to herein as muscarinic acetylcholinereceptor 6 (“mACHR-6”) polypeptide or protein, which is capable of, forexample, modulating the effects of acetylcholine on acetylcholineresponsive cells e.g., by modulating phospholipase C signaling/activity.Nucleic acid molecules encoding an mACHR-6 polypeptide are referred toherein as mACHR-6 nucleic acid molecules. In a preferred embodiment, themACHR-6 polypeptide interacts with (e.g., binds to) a protein which is amember of the G family of proteins. Examples of such proteins includeGo, Gi, Gs, Gq and Gt. These proteins are described in Lodish H. et al.Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y.,1995); Dolphin A. C. et al. (1987) Trends Neurosci. 10:53; andBirnbaumer L. et al. (1992) Cell 71:1069, the contents of which areexpressly incorporated herein by reference.

[0006] In a preferred embodiment, the mACHR-6 polypeptide interacts with(e.g., binds to) acetylcholine. Acetylcholine is the predominantneurotransmitter in the sympathetic and parasympathetic preganglionicsynapses, as well as in the parasympathetic postganglionic synapses andin some sympathetic postganglionic synapses. Synapses in whichacetylcholine is the neurotransmitter are called cholinergic synapses.Acetylcholine acts to regulate smooth muscle contraction, heart rate,glandular function such as gastric acid secretion, and neural functionsuch as release of neurotransmitters from the brain. The mACHR-6polypeptide of the present invention binds to acetylcholine and servesto mediate the acetylcholine induced signal to the cell. Thus, mACHR-6molecules can be used as targets to modulate acetylcholine inducedfunctions and thus to treat disorders associated with, for example,abnormal acetylcholine levels, or abnormal or aberrant mACHR-6polypeptide activity or nucleic acid expression.

[0007] Accordingly, one aspect of the invention pertains to isolatednucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequenceencoding an mACHR-6 polypeptide or biologically active portions thereof,as well as nucleic acid fragments suitable as primers or hybridizationprobes for the detection of mACHR-6-encoding nucleic acid (e.g., mRNA).In particularly preferred embodiments, the isolated nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:1, 4, or 31, thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or the coding region or a complementof either of these nucleotide sequences. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which encodes naturally occurringallelic variants, genetically altered variants and non-human and non-rathomologues of the mACHR-6 polypeptides described herein. Such nucleicacid molecules are identifiable as being able to hybridize to or whichare at least about 30-35%, preferably at least about 40-45%, morepreferably at least about 50-55%, even more preferably at least about60-65%, yet more preferably at least about 70-75%, still more preferablyat least about 80-85%, and most preferably at least about 90-95% or morehomologous to the nucleotide sequence shown in SEQ ID NO:1, 4, or 31,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or a portion of either of thesenucleotide sequences. In other preferred embodiments, the isolatednucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2, 5,or 32 or an amino acid sequence encoded by the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC® as Accession Number______. The preferred mACHR-6 polypeptides of the present invention alsopreferably possess at least one of the mACHR-6 activities describedherein.

[0008] In another embodiment, the isolated nucleic acid molecule encodesa polypeptide or portion thereof wherein the polypeptide or portionthereof includes an amino acid sequence which is sufficiently homologousto an amino acid sequence of SEQ ID NO:2, 5, or 32, e.g., sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, 5, or 32 such thatthe polypeptide or portion thereof maintains an mACHR-6 activity.Preferably, the polypeptide or portion thereof encoded by the nucleicacid molecule maintains the ability to modulate an acetylcholineresponse in an acetylcholine responsive cell. In one embodiment, thepolypeptide encoded by the nucleic acid molecule is at least about30-35%, preferably at least about 40-45%, more preferably at least about50-55%, even more preferably at least about 60-65%, yet more preferablyat least about 70-75%, still more preferably at least about 80-85%, andmost preferably at least about 90-95% or more homologous to the aminoacid sequence of SEQ ID NO:2, 5, or 32 (e.g., the entire amino acidsequence of SEQ ID NO:2, 5, or 32) or the amino acid sequence encoded bythe nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. In another preferred embodiment thenucleic acid molecule encodes a polypeptide fragment comprising at least15 contiguous amino acids of SEQ ID NO:2, 5, or 32. In yet anotherpreferred embodiment, the polypeptide is a full length human-polypeptidewhich is substantially homologous to the entire amino acid sequence ofSEQ ID NO:2, 5, or 32 (encoded by the open reading frame shown in SEQ IDNO:3, 6, or 33, respectively). In still another preferred embodiment,the nucleic acid molecule encodes a naturally occurring allelic variantof the polypeptide of SEQ ID NO:2, 5, or 32 and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, 4, or 31, respectively.

[0009] In yet another embodiment, the isolated nucleic acid molecule isderived from a human and encodes a portion of a polypeptide whichincludes a transmembrane domain. Preferably, the transmembrane domainencoded by the human nucleic acid molecule is at least about 50-55%,preferably at least about 60-65%, more preferably at least about 70-75%,even more preferably at least about 80-85%, and most preferably at leastabout 90-95% or more homologous to any of the human transmembranedomains (i.e., amino acid residues 34-59, 109-130, 152-174, 197-219, or396416) of SEQ ID NO:2 which are shown as separate sequences designatedSEQ ID NOs:7, 9, 10, 11, and 13, respectively, or to any of the rattransmembrane domains (i.e., amino acid residues 34-59, 73-91, 109-130,152-174, 197-219, 360-380, or 396-416 of SEQ ID NO:5 which are shown asseparate sequences designated SEQ ID NOs:14, 15, 16, 17, 18, 19, and 20,respectively or amino acid residues 1-8, 26-47, 69-91, 114-136, 277-297,or 313-333 of SEQ ID NO:32 which are shown as separate sequencesdesignated SEQ ID NOs:34, 35, 36, 37, 38, or 39, respectively). Morepreferably, the transmembrane domain encoded by the human nucleic acidmolecule is at least about 75-80%, preferably at least about 80-85%,more preferably at least about 85-90%, and most preferably at leastabout 90-95% or more homologous to the transmembrane domain (i.e., aminoacid residues 360-380) of SEQ ID NO:2 which is shown as a separatesequence designated SEQ ID NO:12, or at least about 80-85%, morepreferably at least about 85-90%, and most preferably at least about90-95% or more homologous to the transmembrane domain (i.e., amino acidresidues 73-91) of SEQ ID NO:2 which is shown as a separate sequencedesignated SEQ ID NO:8.

[0010] In another preferred embodiment, the isolated nucleic acidmolecule is derived from a human and encodes a polypeptide (e.g., anmACHR-6 fusion polypeptide such as an mACHR-6 polypeptide fused with aheterologous polypeptide) which includes a transmembrane domain which isat least about 75% or more homologous to SEQ ID NO:7-13, or to thecorresponding rat sequences shown as SEQ ID NOs:14-20 and has one ormore of the following mACHR-6 activities: 1) it can interact with (e.g.,bind to) acetylcholine; 2) it can interact with (e.g., bind to) a Gprotein or another protein which naturally binds to mACHR-6; 3) it canmodulate the activity of an ion channel (e.g., a potassium channel or acalcium channel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

[0011] In another embodiment, the isolated nucleic acid molecule is atleast 15 nucleotides, e.g., at least 15 contiguous nucleotides, inlength and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 4, or 31 orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. Preferably, the isolated nucleic acidmolecule corresponds to a naturally-occurring nucleic acid molecule.More preferably, the isolated nucleic acid encodes naturally-occurringhuman mACHR-6 or a biologically active portion thereof. Moreover, giventhe disclosure herein of an mACHR-6-encoding cDNA sequence (e.g., SEQ IDNO:1, 4, or 31), antisense nucleic acid molecules (e.g., molecules whichare complementary to the coding strand of the mACHR-6 cDNA sequence) arealso provided by the invention.

[0012] Another aspect of the invention pertains to vectors, e.g.,recombinant expression vectors, containing the nucleic acid molecules ofthe invention and host cells into which such vectors have beenintroduced. In one embodiment, such a host cell is used to produce anmACHR-6 polypeptide by culturing the host cell in a suitable medium. Ifdesired, the mACHR-6 polypeptide can then be isolated from the medium orthe host cell.

[0013] Yet another aspect of the invention pertains to transgenicnon-human animals in which an mACHR-6 gene has been introduced oraltered. In one embodiment, the genome of the non-human animal has beenaltered by introduction of a nucleic acid molecule of the inventionencoding mACHR-6 as a transgene. In another embodiment, an endogenousmACHR-6 gene within the genome of the non-human animal has been altered,e.g., functionally disrupted, by homologous recombination.

[0014] Still another aspect of the invention pertains to an isolatedmACHR-6 polypeptide or a portion, e.g., a biologically active portion,thereof. In a preferred embodiment, the isolated mACHR-6 polypeptide orportion thereof can modulate an acetylcholine response in anacetylcholine responsive cell. In another preferred embodiment, theisolated mACHR-6 polypeptide or portion thereof is sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, 5, or 32 such thatthe polypeptide or portion thereof maintains the ability to modulate anacetylcholine response in an acetylcholine responsive cell.

[0015] In one embodiment, the biologically active portion of the mACHR-6polypeptide includes a domain or motif, preferably a domain or motifwhich has an mACHR-6 activity. The domain can be transmembrane domain.If the active portion of the polypeptide which comprises thetransmembrane domain is isolated or derived from a human, it ispreferred that the transmembrane domain be at least about 75-80%,preferably at least about 80-85%, more preferably at least about 85-90%,and most preferably at least about 90-95% or more homologous to SEQ IDNO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 34, 35, 36, 37,38, or 39. Preferably, the biologically active portion of the mACHR-6polypeptide which includes a transmembrane domain also has one of thefollowing mACHR-6 activities: 1) it can interact with (e.g., bind to)acetylcholine; 2) it can interact with (e.g., bind to) a G protein oranother protein which naturally binds to mACHR-6; 3) it can modulate theactivity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

[0016] The invention also provides an isolated preparation of an mACHR-6polypeptide. In preferred embodiments, the mACHR-6 polypeptide comprisesthe amino acid sequence of SEQ ID NO:2, 5, or 32 or an amino acidsequence encoded by the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC® as Accession Number ______. In anotherpreferred embodiment, the invention pertains to an isolated full lengthpolypeptide which is substantially homologous to the entire amino acidsequence of SEQ ID NO:2, 5, or 32 (encoded by the open reading frameshown in SEQ ID NO:3, 6, or 33, respectively) such as a naturallyoccurring allelic variant of the mACHR-6 polypeptides described herein.In yet another embodiment, the polypeptide is at least about 30-35%,preferably at least about 40-45%, more preferably at least about 50-55%,even more preferably at least about 60-65%, yet more preferably at leastabout 70-75%, still more preferably at least about 80-85%, and mostpreferably at least about 90-95% or more homologous to the entire aminoacid sequence of SEQ ID NO:2, 5, or 32 such as a non-human or non-rathomologue of the mACHR-6 polypeptides described herein. In otherembodiments, the isolated mACHR-6 polypeptide comprises an amino acidsequence which is at least about 30-40% or more homologous to the aminoacid sequence of SEQ ID NO:2, 5, or 32 and has an one or more of thefollowing mACHR-6 activities: 1) it can interact with (e.g., bind to)acetylcholine; 2) it can interact with (e.g., bind to) a G protein oranother protein which naturally binds to mACHR-6; 3) it can modulate theactivity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

[0017] Alternatively, the isolated mACHR-6 polypeptide can comprise anamino acid sequence which is encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, or is at leastabout 30-35%, preferably at least about 40-45%, more preferably at leastabout 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more homologous tothe nucleotide sequence of SEQ ID NO:1, 4, or 31 or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______, such as the allelic variants and non-human andnon-rat homologues of the mACHR-6 polypeptides described herein as wellas genetically altered variants generated by recombinant DNAmethodologies. It is also preferred that the preferred forms of mACHR-6also have one or more of the mACHR-6 activities described herein.

[0018] The mACHR-6 polypeptide (or protein) or a biologically activeportion thereof can be operatively linked to a non-mACHR-6 polypeptide(e.g., a polypeptide comprising heterologous amino acid sequences) toform a fusion polypeptide. In addition, the mACHR-6 polypeptide or abiologically active portion thereof can be incorporated into apharmaceutical composition comprising the polypeptide and apharmaceutically acceptable carrier.

[0019] The mACHR-6 polypeptide of the invention, or portions orfragments thereof, can be used to prepare anti-mACHR-6 antibodies.Accordingly, the invention also provides an antigenic peptide of mACHR-6which comprises at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO:2, 5, or 32 and encompasses an epitope ofmACHR-6 such that an antibody raised against the peptide forms aspecific immune complex with mACHR-6. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues. Theinvention further provides an antibody that specifically binds mACHR-6.In one embodiment, the antibody is monoclonal. In another embodiment,the antibody is coupled to a detectable substance. In yet anotherembodiment, the antibody is incorporated into a pharmaceuticalcomposition comprising the antibody and a pharmaceutically acceptablecarrier.

[0020] Another aspect of the invention pertains to methods formodulating a cell activity mediated by mACHR-6, e.g., biologicalprocesses mediated by phosphatidylinositol turnover and signaling;secretion of a molecule, e.g., a neurotransmitter from a brain cell, oran enzyme from a gland cell; or contraction of a smooth muscle cell,e.g., an ileum smooth muscle cell or a cardiac cell, e.g., acardiomyocyte. Such methods include contacting the cell with an agentwhich modulates mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression such that an mACHR-6-mediated cell activity is alteredrelative to the same cellular activity which occurs in the absence ofthe agent. In a preferred embodiment, the cell (e.g., a smooth musclecell or a neural cell) is capable of responding to acetylcholine througha signaling pathway involving an mACHR-6 polypeptide. The agent whichmodulates mACHR-6 activity can be an agent which stimulates mACHR-6polypeptide activity or mACHR-6 nucleic acid expression. Examples ofagents which stimulate mACHR-6 polypeptide activity or mACHR-6 nucleicacid expression include small molecules, active mACHR-6 polypeptides,and nucleic acids encoding mACHR-6 that have been introduced into thecell. Examples of agents which inhibit mACHR-6 activity or expressioninclude small molecules, antisense mACHR-6 nucleic acid molecules, andantibodies that specifically bind to mACHR-6. In a preferred embodiment,the cell is present within a subject and the agent is administered tothe subject.

[0021] The present invention also pertains to methods for treatingsubjects having various disorders, e.g., disorders mediated by abnormalmACHR-6 polypeptide activity, such as conditions caused by over, under,or inappropriate expression of mACHR-6. For example, the inventionpertains to methods for treating a subject having a disordercharacterized by aberrant mACHR-6 polypeptide activity or nucleic acidexpression such as a nervous system disorder, e.g., a cognitivedisorder, a sleep disorder, a movement disorder, a schizo-effectivedisorder, a disorder affecting pain generation mechanisms, a drinkingdisorder, or an eating disorder; a smooth muscle related disorder, e.g.,irritable bowel syndrome, a cardiac muscle related disorder, e.g.,bradycardia, or a gland related disorder, e.g., xerostomia. Thesemethods include administering to the subject an mACHR-6 modulator (e.g.,a small molecule) such that treatment of the subject occurs.

[0022] In other embodiments, the invention pertains to methods fortreating a subject having a disorder mediated by abnormal mACHR-6polypeptide activity, such as conditions caused by over, under, orinappropriate expression of mACHR-6, e.g., a nervous system disorder,e.g., a cognitive disorder, a sleep disorder, a movement disorder, aschizo-effective disorder, a disorder affecting pain generationmechanisms, a drinking disorder, or an eating disorder; a smooth musclerelated disorder, e.g., irritable bowel syndrome; a cardiac musclerelated disorder, e.g., bradycardia; or a gland related disorder, e.g.,xerostomia. The method includes administering to the subject an mACHR-6polypeptide or portion thereof such that treatment occurs. A nervoussystem disorder, smooth muscle related disorder, cardiac muscle relateddisorder or a gland related disorder can also be treated according tothe invention by administering to the subject having the disorder anucleic acid encoding an mACHR-6 polypeptide or portion thereof suchthat treatment occurs.

[0023] The invention also pertains to methods for detecting naturallyoccurring and recombinantly created genetic mutations in an mACHR-6gene, thereby determining if a subject with the mutated gene is at riskfor (or is predisposed to have) a disorder characterized by aberrant orabnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity, e.g., a nervous system disorder, a smooth muscle relateddisorder, a cardiac muscle related disorder or a gland related disorder.In preferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic mutationcharacterized by an alteration affecting the integrity of a geneencoding an mACHR-6 polypeptide, or the misexpression of the mACHR-6gene, such as that caused by a nucleic acid base substitution, deletionor addition, or gross sequence changes caused by a genetic translation,inversion or insertion.

[0024] Another aspect of the invention pertains to methods for detectingthe presence of mACHR-6, or allelic variants thereof, in a biologicalsample. In a preferred embodiment, the methods involve contacting abiological sample (e.g., a brain or smooth muscle cell sample) with acompound or an agent capable of detecting mACHR-6 polypeptide or mACHR-6mRNA such that the presence of mACHR-6 is detected in the biologicalsample. The compound or agent can be, for example, a labeled orlabelable nucleic acid probe capable of hybridizing to mACHR-6 mRNA or alabeled or labelable antibody capable of binding to mACHR-6 polypeptide.The invention further provides methods for diagnosis of a subject with,for example, a nervous system disorder, a smooth muscle relateddisorder, a cardiac muscle related disorder or a gland related disorder,based on detection of mACHR-6 polypeptide or mRNA. In one embodiment,the method involves contacting a cell or tissue sample (e.g., a brain orsmooth muscle cell sample) from the subject with an agent capable ofdetecting mACHR-6 polypeptide or mRNA, determining the amount of mACHR-6polypeptide or mRNA expressed in the cell or tissue sample, comparingthe amount of mACHR-6 polypeptide or mRNA expressed in the cell ortissue sample to a control sample and forming a diagnosis based on theamount of mACHR-6 polypeptide or mRNA expressed in the cell or tissuesample as compared to the control sample. Preferably, the cell sample isa brain cell sample. Kits for detecting mACHR-6 in a biological samplewhich include agents capable of detecting mACHR-6 polypeptide or mRNAare also within the scope of the invention.

[0025] Still another aspect of the invention pertains to methods, e.g.,screening assays, for identifying a compound, e.g., a test compound, fortreating a disorder characterized by aberrant mACHR-6 nucleic acidexpression or polypeptide activity, e.g., a nervous system disorder, asmooth muscle related disorder, a cardiac muscle related disorder or agland related disorder. These methods typically include assaying theability of the compound or agent to modulate the expression of themACHR-6 gene or the activity of the mACHR-6 polypeptide therebyidentifying a compound for treating a disorder characterized by aberrantmACHR-6 nucleic acid expression or polypeptide activity. In a preferredembodiment, the method involves contacting a biological sample, e.g., acell or tissue sample, e.g., a brain or smooth muscle cell sample,obtained from a subject having the disorder with the compound or agent,determining the amount of mACHR-6 polypeptide expressed and/or measuringthe activity of the mACHR-6 polypeptide in the biological sample,comparing the amount of mACHR-6 polypeptide expressed in the biologicalsample and/or the measurable mACHR-6 biological activity in the cell tothat of a control sample. An alteration in the amount of mACHR-6polypeptide expression or mACHR-6 activity in the cell exposed to thecompound or agent in comparison to the control is indicative of amodulation of mACHR-6 expression and/or mACHR-6 activity.

[0026] The invention also pertains to methods for identifying a compoundor agent, e.g., a test compound or agent, which interacts with (e.g.,binds to) an mACHR-6 polypeptide. These methods can include the steps ofcontacting the mACHR-6 polypeptide with the compound or agent underconditions which allow binding of the compound to the mACHR-6polypeptide to form a complex and detecting the formation of a complexof the mACHR-6 polypeptide and the compound in which the ability of thecompound to bind to the mACHR-6 polypeptide is indicated by the presenceof the compound in the complex.

[0027] The invention further pertains to methods for identifying acompound or agent, e.g., a test compound or agent, which modulates,e.g., stimulates or inhibits, the interaction of the mACHR-6 polypeptidewith a target molecule, e.g., acetylcholine, or a cellular proteininvolved in phosphatidylinositol turnover and signaling. In thesemethods, the mACHR-6 polypeptide is contacted, in the presence of thecompound or agent, with the target molecule under conditions which allowbinding of the target molecule to the mACHR-6 polypeptide to form acomplex. An alteration, e.g., an increase or decrease, in complexformation between the mACHR-6 polypeptide and the target molecule ascompared to the amount of complex formed in the absence of the compoundor agent is indicative of the ability of the compound or agent tomodulate the interaction of the mACHR-6 polypeptide with a targetmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 depicts the human mACHR-6 nucleotide (SEQ ID NO:1) andamino acid (SEQ ID NO:2) sequences. The coding region without the 5′ and3′ untranslated region of the human mACHR-6 gene is shown in SEQ IDNO:3.

[0029]FIG. 2 depicts the rat mACHR-6 nucleotide (SEQ ID NO:4) and aminoacid (SEQ ID NO:5) sequences. The coding region without the 5′0 and 3′untranslated region of the rat mACHR-6 gene is shown in SEQ ID NO:6.

[0030]FIG. 3 depicts the partial rat mACHR-6 nucleotide (SEQ ID NO:31.)and amino acid (SEQ ID NO:32) sequences. The partial coding regionwithout the 3′ untranslated region of the rat mACHR-6 gene is shown inSEQ ID NO:33.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based on the discovery of novelmolecules, referred to herein as mACHR-6 nucleic acid and polypeptidemolecules, which play a role in or function in acetylcholine signalingpathways. In one embodiment, the mACHR-6 molecules modulate the activityof one or more proteins involved in a neurotransmitter signalingpathway, e.g., an acetylcholine signaling pathway. In a preferredembodiment, the mACHR-6 molecules of the present invention are capableof modulating the activity of proteins involved in the acetylcholinesignaling pathway to thereby modulate the effects of acetylcholine onacetylcholine responsive cells.

[0032] As used herein, the phrase “acetylcholine responsive cells”refers to cells which have a function which can be modulated (e.g.,stimulated or inhibited) by the neurotransmitter acetylcholine. Examplesof such functions include mobilization of intracellular molecules whichparticipate in a signal transduction pathway, e.g., phosphatidylinositol4,5-bisphosphate (PIP₂) or inositol 1,4,5-triphosphate (IP₃),polarization of the plasma membrane, production or secretion ofmolecules, alteration-in the structure of a cellular component, cellproliferation, cell migration, cell differentiation, and cell survival.Acetylcholine responsive cells preferably express an acetylcholinereceptor, e.g., a muscarinic receptor. Examples of acetylcholineresponsive cells include neural cells, e.g., central nervous system andperipheral nervous system cells (such as sympathetic and parasympatheticneurons); smooth muscle cells, e.g., smooth muscle cells in thedigestive tract, the urinary tract, the blood vessels, the airways andthe lungs, or the uterus; cardiac muscle cells, e.g., cardiomyocytes;and gland cells such as exocrine gland cells, e.g., pancreatic glandcells, e.g., pancreatic beta cells, tear gland cells, sweat gland cells,or parotid gland cells.

[0033] Depending on the type of cell, the response elicited byacetylcholine is different. For example, in neural cells, acetylcholineregulates ion channels, and neural signal to noise ratio. Inhibition orover stimulation of the activity of proteins involved in theacetylcholine signaling pathway or misexpression of acetylcholine canlead to hypo- or hyperpolarization of the neural plasma membrane and toperturbed neural signal to noise ratio, which can in turn lead tonervous system related disorders. Examples of nervous system relateddisorders include cognitive disorders, e.g., memory and learningdisorders, such as amnesia, apraxia, agnosia, amnestic dysnomia,amnestic spatial disorientation, Kluver-Bucy syndrome, Alzheimer'srelated memory loss (Eglen R. M. (1996) Pharmacol. and Toxicol.78(2):59-68; Perry E. K. (1995) Brain and Cognition 28(3):240-58) andlearning disability; disorders affecting consciousness, e.g., visualhallucinations, perceptual disturbances, or delerium associated withLewy body dementia; schitzo-effective disorders (Dean B. (1996) Mol.Psychiatry 1(1):54-8), schizophrenia with mood swings (Bymaster F. P.(1997) J. Clin. Psychiatry 58 (suppl. 10):28-36; Yeomans J. S. (1995)Neuropharmacol. 12(1):3-16; Reimann D. (1994) J. Psychiatric Res.28(3):195-210), depressive illness (primary or secondary); affectivedisorders (Janowsky D. S. (1994) Am. J. Med. Genetics 54(4):335-44);sleep disorders (Kimura F. (1997) J. Neurophysiol. 77(2):709-16), e.g.,REM sleep abnormalities in patients suffering from, for example,depression (Riemann D. (1994) J. Psychosomatic Res. 38 Suppl. 1:15-25;Bourgin P. (1995) Neuroreport 6(3): 532-6), paradoxical sleepabnormalities (Sakai K. (1997) Eur. J. Neuroscience 9(3):415-23),sleep-wakefulness, and body temperature or respiratory depressionabnormalities during sleep (Shuman S. L. (1995) Am. J. Physiol. 269(2 Pt2):R308-17; Mallick B. N. (1997) Brain Res. 750(1-2):311-7). Otherexamples of nervous system related disorders include disorders affectingpain generation mechanisms, e.g., pain related to irritable bowelsyndrome (Mitch C. H. (1997) J. Med. Chem. 40(4):538-46; Shannon H. E.(1997) J. Pharmac. and Exp. Therapeutics 281(2):884-94; Bouaziz H.(1995) Anesthesia and Analgesia 80(6):1140-4; or Guimaraes A. P. (1994)Brain Res. 647(2):220-30) or chest pain; movement disorders (Monassi C.R. (1997) Physiol. and Behav. 62(1):53-9), e.g., Parkinson's diseaserelated movement disorders (Finn M. (1997) Pharmacol. Biochem. &Behavior 57(1-2):243-9; Mayorga A. J. (1997) Pharmacol. Biochem. &Behavior 56(2):273-9); eating disorders, e.g., insulin hypersecretionrelated obesity (Maccario M. (1997) J. Endocrinol. Invest. 20(1):8-12;Premawardhana L. D. (1994) Clin. Endocrinol. 40(5): 617-21); or drinkingdisorders, e.g., diabetic polydipsia (Murzi E. (1997) Brain Res.752(1-2):184-8; Yang X. (1994) Pharmacol. Biochem. & Behavior49(1):1-6).

[0034] In smooth muscle, acetylcholine regulates (e.g., stimulates orinhibits) contraction. Inhibition or overstimulation of the activity ofproteins involved in the acetylcholine signaling pathway ormisexpression of acetylcholine can lead to smooth muscle relateddisorders such as irritable bowel syndrome, diverticular disease,urinary incontinence, oesophageal achalasia, or chronic obstructiveairways disease.

[0035] In cardiac muscle, acetylcholine induces a reduction in the heartrate and in cardiac contractility. Inhibition or overstimulation of theactivity of proteins involved in the acetylcholine signaling pathway ormisexpression of acetylcholine can lead to heart muscle relateddisorders such as pathologic bradycardia or tachycardia, arrhythmia,flutter or fibrillation.

[0036] In glands such as exocrine glands, acetylcholine regulates thesecretion of enzymes or hormones, e.g., in the parotid glandacetylcholine induces the release of amylase, and in the pancreasacetylcholine induces the release of digestive enzymes and insulin.Inhibition or over stimulation of the activity of proteins involved inthe acetylcholine signaling pathway or misexpression of acetylcholinecan lead to gland related disorders such as xerostomia, or diabetesmellitus.

[0037] In a particularly preferred embodiment, the mACHR-6 molecules arecapable of modulating the activity of G proteins, as well asphosphatidylinositol metabolism and turnover in acetylcholine responsivecells. As used herein, a “G protein” is a protein which participates, asa secondary signal, in a variety of intracellular signal transductionpathways, e.g., in the acetylcholine signaling pathway primarily throughphosphatidylinositol metabolism and turnover. G proteins represent afamily of heterotrimeric proteins composed of α, β and γ subunits, whichbind guanine nucleotides. These proteins are usually linked to cellsurface receptors, e.g., receptors containing seven transmembranedomains, such as the muscarinic receptors. Following ligand binding tothe receptor, a conformational change is transmitted to the G protein,which causes the α-subunit to exchange a bound GDP molecule for a GTPmolecule and to dissociate from the βγ-subunits. The GTP-bound form ofthe α-subunit typically functions as an effector-modulating moiety,leading to the production of second messengers, such as cyclic AMP(e.g., by activation of adenylate cyclase), diacylglycerol or inositolphosphates. Greater than 20 different types of α-subunits are known inman, which associate with a smaller pool of β and γ subunits. Examplesof mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins aredescribed extensively in Lodish H. et al. Molecular Cell Biology,(Scientific American Books Inc., New York, N.Y., 1995).

[0038] As used herein, “phosphatidylinositol turnover and metabolism”refers to the molecules involved in the turnover and metabolism ofphosphatidylinositol 4,5-bisphosphate (PIP₂) as well as to theactivities of these molecules. PIP₂ is a phospholipid found in thecytosolic leaflet of the plasma membrane. Binding of acetylcholine to amuscarinic receptor activates the plasma-membrane enzyme phospholipase Cwhich in turn can hydrolyze PIP₂ to produce 1,2-diacylglycerol (DAG) andinositol 1,4,5-triphosphate (IP₃). Once formed IP₃ can diffuse to theendoplasmic reticulum surface where it can bind an IP₃ receptor, e.g., acalcium channel protein containing an IP₃ binding site. IP₃ binding caninduce opening of the channel, allowing calcium ions to be released intothe cytoplasm. 1P₃ can also be phosphorylated by a specific kinase toform inositol 1,3,4,5-tetraphosphate (IP₄), a molecule which can causecalcium entry into the cytoplasm from the extracellular medium. IP₃ andIP₄ can subsequently be hydrolyzed very rapidly to the inactive productsinositol 1,4-biphosphate (IP₂) and inositol 1,3,4-triphosphate,respectively. These inactive products can be recycled by the cell tosynthesize PIP₂. The other second messenger produced by the hydrolysisof PIP₂, namely 1,2-diacylglycerol (DAG), remains in the cell membranewhere it can serve to activate the enzyme protein kinase C. Proteinkinase C is usually found soluble in the cytoplasm of the cell, but uponan increase in the intracellular calcium concentration, this enzyme canmove to the plasma membrane where it can be activated by DAG. Theactivation of protein kinase C in different cells results in variouscellular responses such as the phosphorylation of glycogen synthase, orthe phosphorylation of various transcription factors, e.g., NF-κB. Thelanguage “phosphatidylinositol activity”, as used herein, refers to anactivity of PIP₂ or one of its metabolites.

[0039] mACHR-6 nucleic acid molecules were identified by screeningappropriate cDNA libraries (described in detail in Example 1). The ratmACHR-6 nucleic acid molecule was identified by screening a rat braincDNA library. Positive clones were sequenced and the partial sequenceswere analyzed by comparison with sequences in a nucleic acid sequencedata base. This analysis indicated that the sequences were homologous tothe muscarinic family of receptors. A longer rat clone was then isolatedand sequenced. The human mACHR-6 nucleic acid molecule was identified byscreening a human cerebellum cDNA library using probes designed based onthe rat sequence.

[0040] Because of its ability to interact with (e.g., bind to)acetylcholine, G proteins and other proteins involved in theacetylcholine signaling pathway, the mACHR-6 polypeptide is also apolypeptide which functions in the acetylcholine signaling pathway.

[0041] The nucleotide sequence of the isolated human mACHR-6 cDNA andthe predicted amino acid sequence of the human mACHR-6 polypeptide areshown in FIG. 1 and in SEQ ID NOs:1 and 2, respectively. A plasmidcontaining the full length nucleotide sequence encoding human mACHR-6was deposited with ATCC® on ______ and assigned Accession Number ______.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

[0042] The nucleotide sequence of the isolated rat mACHR-6 cDNA and thepredicted amino acid sequence of the rat mACHR-6 polypeptide are shownin FIG. 2 and in SEQ ID NOs:4 and 5, respectively. A plasmid containingthe full length nucleotide sequence encoding rat mACHR-6 was depositedwith ATCC® on and assigned Accession Number ______. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

[0043] The nucleotide sequence of the isolated partial rat mACHR-6 cDNAand the predicted amino acid sequence of the partial rat mACHR-6polypeptide are shown in FIG. 3 and in SEQ ID NOs:31 and 32,respectively.

[0044] The human mACHR-6 gene, which is approximately 2689 nucleotidesin length, encodes a full length polypeptide having a molecular weightof approximately 51.2 KDa and which is approximately 445 amino acidresidues in length. The human mACHR-6 polypeptide is expressed at leastin the brain, in particular, regions of the brain such as thecerebellum, the cerebral cortex, the medulla, the occipital pole, thefrontal lobe, the temporal lobe, the putamen, the corpus callosum theamygdala, the caudate nucleus, the hippocampus, the substantia nigra,the subthalamic nucleus and the thalamus; spinal cord, placenta, lungs,spleen, liver, skeletal muscle, kidney, and testis. Based on structuralanalysis, amino acid residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8),109-130 (SEQ ID NO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11),360-380 (SEQ ID NO:12), and 396416 (SEQ ID NO:13) comprise transmembranedomains. As used herein, the term “transmembrane domain” refers to astructural amino acid motif which includes a hydrophobic helix thatspans the plasma membrane. A transmembrane domain also preferablyincludes a series of conserved serine, threonine, and tyrosine residues.For example, the transmembrane domains between residues 109-130 (SEQ IDNO:9), 197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and 396416 (SEQID NO:13), contain threonine and tyrosine residues (located about 1-2helical turns away from the membrane surface), which are important forligand, e.g., acetylcholine, binding. Other important residues in thetransmembrane domains include the conserved aspartate residue in thetransmembrane domain between residues 109-130 (SEQ ID NO:9) and theconserved proline residue in the transmembrane domain between residues152-174 (SEQ ID NO:10), which are also important for ligand, e.g.,acetylcholine, binding. A skilled artisan will readily appreciate thatthe beginning and ending amino acid residue recited for variousdomains/fragments of mACHR-6 are based on structural analysis and thatthe actual beginning/ending amino acid for each may vary by a few aminoacids from that identified herein.

[0045] The rat mACHR-6 gene, which is approximately 3244 nucleotides inlength, encodes a full length polypeptide having a molecular weight ofapproximately 51.2 kDa and which is at least about 445 amino acidresidues in length. The rat mACHR-6 polypeptide is expressed in thebrain. Amino acid residues 34-59 (SEQ ID NO:14), 73-91 (SEQ ID NO:15),109-130 (SEQ ID NO:16), 152-174 (SEQ ID NO:17), 197-219 (SEQ ID NO:18),360-380 (SEQ ID NO:19) and 396-416 (SEQ ID NO:20) comprise transmembranedomains.

[0046] The rat mACHR-6 gene, which is at least about 2218 nucleotides inlength, encodes a full length polypeptide having a molecular weight ofat least about 41.6 kDa and which is at least about 362 amino acidresidues in length. The rat mACHR-6 polypeptide is expressed in thebrain. Amino acid residues 1-8 (SEQ ID NO:14), 26-47 (SEQ ID NO:15),69-91 (SEQ ID NO:16), 114-136 (SEQ ID NO:17), 277-297 (SEQ ID NO:18),and 313-333 (SEQ ID NO:19) comprise transmembrane domains.

[0047] The partial rat mACHR-6 gene, which is at least about 2218nucleotides in length, encodes a polypeptide having a molecular weightof at least about 41.6 kDa and which is at least about 362 amino acidresidues in length. The rat mACHR-6 polypeptide is expressed in thebrain. Amino acid residues 1-8 (SEQ ID NO:34), 26-47 (SEQ ID NO:35),69-91 (SEQ ID NO:36), 114-136 (SEQ ID NO:37), 277-297 (SEQ ID NO:38),and 313-333 (SEQ ID NO:39) comprise transmembrane domains.

[0048] The mACHR-6 polypeptide, a biologically active portion orfragment of the polypeptide, or an allelic variant thereof can have oneor more of the following mACHR-6 activities: 1) it can interact with(e.g., bind to) acetylcholine; 2) it can interact with (e.g., bind to) aG protein or another protein which naturally binds to mACHR-6; 3) it canmodulate the activity of an ion channel (e.g., a potassium channel or acalcium channel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

[0049] Various aspects of the invention are described in further detailin the following subsections:

[0050] I. Isolated Nucleic Acid Molecules

[0051] One aspect of the invention pertains to isolated nucleic acidmolecules that encode mACHR-6 or biologically active portions thereof,as well as nucleic acid fragments sufficient for use as hybridizationprobes to identify mACHR-6-encoding nucleic acid (e.g., mACHR-6 mRNA).As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatedmACHR-6 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived (e.g., a hippocampal cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

[0052] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, 4, or 31,or a portion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example, ahuman mACHR-6 cDNA can be isolated from a human hippocampus libraryusing all or portion of SEQ ID NO:1, 4, or 31 as a hybridization probeand standard hybridization techniques (e.g., as described in Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleicacid molecule encompassing all or a portion of SEQ ID NO:1, 4, or 31 canbe isolated by the polymerase chain reaction using oligonucleotideprimers designed based upon the sequence of SEQ ID NO:1, 4, or 31. Forexample, mRNA can be isolated from normal brain cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979)Biochemistry 18: 5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO:1, 4, or 31. A nucleic acidof the invention can be amplified using cDNA or, alternatively, genomicDNA, as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to an mACHR-6nucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

[0053] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:1, 4,or 31 or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC® as Accession Number ______. The sequence of SEQ IDNO:1 corresponds to the human mACHR-6 cDNA. This cDNA comprisessequences encoding the human mACHR-6 polypeptide (i.e., “the codingregion”, from nucleotides 291 to 1628 of SEQ ID NO:1), as well as 5′untranslated sequences (nucleotides 1 to 290 of SEQ ID NO:1) and 3′untranslated sequences (nucleotides 1629 to 2689 of SEQ ID NO:1).Alternatively, the nucleic acid molecule can comprise only the codingregion of SEQ ID NO:1 (e.g., nucleotides 291 to 1628 of SEQ ID NO:1,shown separately as SEQ ID NO:3). The sequence of SEQ ID NO:4corresponds to the rat mACHR-6 cDNA. This cDNA comprises sequencesencoding the rat mACHR-6 polypeptide (i.e., “the coding region”, fromnucleotides 778 to 2112 of SEQ ID NO:4), as well as 5′ untranslatedsequences (nucleotides 1 to 777 of SEQ ID NO:4), and 3′ untranslatedsequences (nucleotides 2113 to 3244 of SEQ ID NO:4). Alternatively, thenucleic acid molecule can comprise only the coding region of SEQ ID NO:4(e.g., nucleotides 778 to 2112 of SEQ ID NO:4, shown separately as SEQID NO:6). The sequence of SEQ ID NO:31 corresponds to the partial ratmACHR-6 cDNA. This cDNA comprises sequences encoding part of the ratmACHR-6 polypeptide (i.e., part of “the coding region”, from nucleotides1 to 1089 of SEQ ID NO:31), and 3′ untranslated sequences (nucleotides1090 to 2218 of SEQ ID NO:31). Alternatively, the nucleic acid moleculecan comprise only the partial coding region of SEQ ID NO:31 (e.g.,nucleotides 1 to 1089, shown separately as SEQ ID NO:33).

[0054] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO:1, 4, or 31,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or a portion of either of thesenucleotide sequences. A nucleic acid molecule which is complementary tothe nucleotide sequence shown in SEQ ID NO:1, 4, or 31 is one which issufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1, 4, or 31 such that it can hybridize to the nucleotide sequenceshown in SEQ ID NO:1, 4, or 31, respectively, thereby forming a stableduplex.

[0055] In still another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence which is atleast about 30-35%, preferably at least about 40-45%, more preferably atleast about 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:1, 4, or 31, or to thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or a portion of these nucleotidesequences. Preferably, such nucleic acid molecules encode functionallyactive or inactive allelic variants of mACHR-6. In an additionalpreferred embodiment, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to the nucleotide sequence shown in SEQ ID NO:1,4, or 31, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC® as Accession Number ______, or a portion of eitherof these nucleotide sequences.

[0056] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the coding region of SEQ ID NO:1, 4, or 31, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of mACHR-6. The nucleotidesequence determined from the cloning of the mACHR-6 gene from a mammalallows for the generation of probes and primers designed for use inidentifying and/or cloning mACHR-6 homologues in other cell types, e.g.,from other tissues, as well as mACHR-6 homologues from other mammals.The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of SEQ ID NO:1, 4, or 31 sense, an anti-sensesequence of SEQ ID NO:1, 4, or 31, or naturally occurring mutantsthereof. Primers based on the nucleotide sequence in SEQ ID NO:1, 4, or31 can be used in PCR reactions to clone mACHR-6 homologues. Probesbased on the mACHR-6 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologouspolypeptides. In preferred embodiments, the probe further comprises alabel group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress an mACHR-6 polypeptide,such as by measuring a level of an mACHR-6-encoding nucleic acid in asample of cells from a subject e.g., detecting mACHR-6 mRNA levels ordetermining whether a genomic mACHR-6 gene has been mutated or deleted.

[0057] In one embodiment, the nucleic acid molecule of the inventionencodes a polypeptide or portion thereof which includes an amino acidsequence which is sufficiently homologous to an amino acid sequence ofSEQ ID NO:2, 5, or 32 or an amino acid sequence encoded by thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______ such that the polypeptide or portionthereof maintains the ability to modulate an acetylcholine response inan acetylcholine responsive cell (e.g., naturally occurring allelicvariants of the rat and human mACHR-6 polypeptides described herein). Asused herein, the language “sufficiently homologous” refers topolypeptides or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO:2, 5, or 32) amino acid residues to an amino acid sequence of SEQID NO:2, 5, or 32 or an amino acid sequence encoded by the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______ such that the polypeptide or portion thereof isable to modulate an acetylcholine response in an acetylcholineresponsive cell or a skilled artisan would clearly recognize it as anon-functional allelic variant of the rat and human mACHR-6 polypeptidesdescribed herein. Acetylcholine, as described herein, initiates avariety of responses in many different cell types. Examples of suchresponses are also described herein. In another embodiment, thepolypeptide is at least about 30-35%, preferably at least about 4045%,more preferably at least about 50-55%, even more preferably at leastabout 60-65%, yet more preferably at least about 70-75%, still morepreferably at least about 80-85%, and most preferably at least about90-95% or more homologous to the amino acid sequence of SEQ ID NO:2, 5,or 32.

[0058] Portions of polypeptides encoded by the mACHR-6 nucleic acidmolecule of the invention are preferably biologically active portions ofthe mACHR-6 polypeptide. As used herein, the term “biologically activeportion of mACHR-6” is intended to include a portion, e.g., adomain/motif, of mACHR-6 that has one or more of the following mACHR-6activities: 1) it can interact with (e.g., bind to) acetylcholine; 2) itcan interact with (e.g., bind to) a G protein or another protein whichnaturally binds to mACHR-6; 3) it can modulate the activity of an ionchannel (e.g., a potassium channel or a calcium channel); 4) it canmodulate cytosolic ion, e.g., calcium, concentration; 5) it can modulatethe release of a neurotransmitter, e.g., acetylcholine, from a neuron,e.g., a presynaptic neuron; 6) it can modulate an acetylcholine responsein an acetylcholine responsive cell (e.g., a smooth muscle cell or agland cell) to, for example, beneficially affect the acetylcholineresponsive cell, e.g., a neuron; 7) it can signal ligand binding viaphosphatidylinositol turnover; and 8) it can modulate, e.g., activate orinhibit, phospholipase C activity.

[0059] Standard binding assays, e.g., immunoprecipitations and yeasttwo-hybrid assays as described herein, can be performed to determine theability of an mACHR-6 polypeptide or a biologically active portionthereof to interact with (e.g., bind to) a binding partner such as a Gprotein. To determine whether an mACHR-6 polypeptide or a biologicallyactive portion thereof can modulate an acetylcholine response in anacetylcholine responsive cell, such cells can be transfected with aconstruct driving the overexpression of an mACHR-6 polypeptide or abiologically active portion thereof. Methods for the preparation ofacetylcholine responsive cells, e.g., intact smooth muscle cells orextracts from such cells are known in the art and described in Glukhovaet al. (1987) Tissue Cell 19 (5):657-63, Childs et al. (1992) J. Biol.Chem. 267 (32):22853-9, and White et al. (1996) J. Biol. Chem. 271(25):15008-17. The cells can be subsequently treated with acetylcholine,and a biological effect of acetylcholine on the cells, such asphosphatidylinositol turnover or cytosolic calcium concentration can bemeasured using methods known in the art (see Hartzell H. C. et al.(1988) Prog. Biophys. Mol. Biol. 52:165-247). Alternatively, transgenicanimals, e.g., mice overexpressing an mACHR-6 polypeptide or abiologically active portion thereof, can be used. Tissues from suchanimals can be obtained and treated with acetylcholine. For example,methods for preparing detergent-skinned muscle fiber bundles are knownin the art (Strauss et al. (1992) Am. J. Physiol. 262:1437-45). Thecontractility of these tissues in response to acetylcholine can bedetermined using, for example, isometric force measurements as describedin Strauss et al., supra. Similarly, to determine whether an mACHR-6polypeptide or a biologically active portion thereof can modulate anacetylcholine response in an acetylcholine responsive cell such as agland cell, gland cells, e.g., parotid gland cells grown in tissueculture, can be transfected with a construct driving the overexpressionof an mACHR-6 polypeptide or a biologically active portion thereof. Thecells can be subsequently treated with acetylcholine, and the effect ofthe acetylcholine on amylase secretion from such cells can be determinedusing, for example an enzymatic assay with a labeled substrate. Thepreferred assays used for mACHR-6 activity will be based onphosphatidylinositol turnover such as those developed for the M1, M3 andM5 classes of receptors (see E. Watson et al. The G Protein LinkedReceptor: FactsBook (Academic Press, Boston, Mass., 1994), the contentsof which are incorporated herein by reference).

[0060] In one embodiment, the biologically active portion of mACHR-6comprises a transmembrane domain. Preferably, the transmembrane domainis encoded by a nucleic acid molecule derived from a human and is atleast about 50-55%, preferably at least about 60-65%, more preferably atleast about 70-75%, even more preferably at least about 80-85%, and mostpreferably at least about 90-95% or more homologous to any of thetransmembrane domains (i.e., amino acid residues 34-59, 109-130,152-174, 197-219, or 396-416) of SEQ ID NO:2 which are shown as separatesequences designated SEQ ID NOs:7, 9, 10, 11, and 13, respectively, orto the rat transmembrane domains (i.e., amino acid residues 34-59,73-91, 109-130, 152-174, 197-219, 360-380, or 396416 of SEQ ID NO:5which are shown as separate sequences designated SEQ ID NOs:14, 15, 16,17, 18, 19, and 20, respectively or amino acid residues 1-8, 2647,69-91, 114-136, 277-297, or 313-333 of SEQ ID NO:32 which are shown asseparate sequences designated SEQ ID NOs:34, 35, 36, 37, 38, or 39,respectively). More preferably, the transmembrane domain encoded by thehuman nucleic acid molecule is at least about 75-80%, preferably atleast about 80-85%, more preferably at least about 85-90%, and mostpreferably at least about 90-95% or more homologous to the transmembranedomain (i.e., amino acid residues 360-380) of SEQ ID NO:2 which is shownas a separate sequence designated SEQ ID NO:12, or at least about80-85%, more preferably at least about 85-90%, and most preferably atleast about 90-95% or more homologous to the transmembrane domain (i.e.,amino acid residues 73-91) of SEQ ID NO:2 which is shown as a separatesequence designated SEQ ID NO:8. In a preferred embodiment, thebiologically active portion of the polypeptide which includes thetransmembrane domain can modulate the activity of a G protein or otherbinding partner in a cell and/or modulate an acetylcholine response inan acetylcholine responsive cell, e.g., a brain cell, to therebybeneficially affect the cell. In a preferred embodiment, thebiologically active portion comprises a transmembrane domain of thehuman mACHR-6 as represented by amino acid residues 34-59 (SEQ ID NO:7),73-91 (SEQ ID NO:8), 109-130 (SEQ ID NO:9),152-174 (SEQ ID NO:10),197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and 396416 (SEQ IDNO:13), a transmembrane domain of the full length rat mACHR-6 asrepresented by amino acid residues 34-59 (SEQ ID NO:14), 73-91 (SEQ IDNO:15), 109-130 (SEQ ID NO:16), 152-174 (SEQ ID NO:17), 197-219 (SEQ IDNO:18), 360-380 (SEQ ID NO:19), and 396-416 (SEQ ID NO:20), or atransmembrane domain of the partial rat mACHR-6 as represented by aminoresidues 1-8 (SEQ ID NO:34),2647 (SEQ ID NO:35),69-91 (SEQ IDNO:36),114-136 (SEQ ID NO:37),277-297 (SEQ ID NO:38), and 313-333 (SEQID NO:39). Additional nucleic acid fragments encoding biologicallyactive portions of mACHR-6 can be prepared by isolating a portion of SEQID NO:1, 4, or 31, expressing the encoded portion of mACHR-6 polypeptideor peptide (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of mACHR-6 polypeptide or peptide.

[0061] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:1, 4, or 31 (andportions thereof) due to degeneracy of the genetic code and thus encodethe same mACHR-6 polypeptide as that encoded by the nucleotide sequenceshown in SEQ ID NO:1, 4, or 31. In another embodiment, an isolatednucleic acid molecule of the invention has a nucleotide sequenceencoding a polypeptide having an amino acid sequence shown in SEQ IDNO:2, 5, or 32 or a polypeptide having an amino acid sequence encoded bythe nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. In a still further embodiment, thenucleic acid molecule of the invention encodes a full length humanpolypeptide which is substantially homologous to the amino acid sequenceof SEQ ID NO:2 or 4 (encoded by the open reading frame shown in SEQ IDNO:3, 6, or 33, respectively) or an amino acid sequence encoded by thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______.

[0062] In addition to the mACHR-6 nucleotide sequence shown in SEQ IDNO:1, 4, or 31, it will be appreciated by those skilled in the art thatDNA sequence polymorphisms that lead to changes in the amino acidsequences of mACHR-6 may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the mACHR-6 gene may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an mACHR-6polypeptide, preferably a mammalian mACHR-6 polypeptide. Such naturalallelic variations can typically result in 1-5% variance in thenucleotide sequence of the mACHR-6 gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in mACHR-6 that arethe result of natural allelic variation are intended to be within thescope of the invention. Such allelic variation includes both activeallelic variants as well as non-active or reduced activity allelicvariants, the later two types typically giving rise to a pathologicaldisorder. Moreover, nucleic acid molecules encoding mACHR-6 polypeptidesfrom other species, and thus which have a nucleotide sequence whichdiffers from the human sequence of SEQ ID NO:1, are intended to bewithin the scope of the invention. Nucleic acid molecules correspondingto natural allelic variants and non-human homologues of the humanmACHR-6 cDNA of the invention can be isolated based on their homology tothe human mACHR-6 nucleic acid disclosed herein using the human cDNA, ora portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions.Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC® as Accession Number ______.In other embodiments, the nucleic acid is at least 30, 50, 100, 250,300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Asused herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural polypeptide). In oneembodiment, the nucleic acid encodes a natural human mACHR-6.

[0063] In addition to naturally-occurring allelic variants of themACHR-6 sequence that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequence of SEQ ID NO:1, 4, or 31, thereby leading tochanges in the amino acid sequence of the encoded mACHR-6 polypeptide,without altering the functional ability of the mACHR-6 polypeptide. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, 4, or 31. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of mACHR-6 (e.g., thesequence of SEQ ID NO:2, 5, or 32) without altering the activity ofmACHR-6, whereas an “essential” amino acid residue is required formACHR-6 activity. For example, conserved amino acid residues, e.g.,aspartates, prolines threonines and tyrosines, in the transmembranedomains of mACHR-6 are most likely important for binding toacetylcholine and are thus essential residues of mACHR-6. Other aminoacid residues, however, (e.g., those that are not conserved or onlysemi-conserved in the transmembrane domain) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering mACHR-6 activity.

[0064] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding mACHR-6 polypeptides that contain changes inamino acid residues that are not essential for mACHR-6 activity. SuchmACHR-6 polypeptides differ in amino acid sequence from SEQ ID NO:2, 5,or 32 yet retain at least one of the mACHR-6 activities describedherein. In one embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence at least about 30-35%, preferably atleast about 40-45%, more preferably at least about 50-55%, even morepreferably at least about 60-65%, yet more preferably at least about70-75%, still more preferably at least about 80-85%, and most preferablyat least about 90-95% or more homologous to the amino acid sequence ofSEQ ID NO:2, 5, or 32.

[0065] To determine the percent homology of two amino acid sequences(e.g., SEQ ID NO:2, 5, or 32 and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one polypeptide ornucleic acid for optimal alignment with the other polypeptide or nucleicacid). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in one sequence (e.g., SEQ ID NO:2, 5, or 32) is occupied bythe same amino acid residue or nucleotide as the corresponding positionin the other sequence (e.g., a mutant form of mACHR-6), then themolecules are homologous at that position (i.e., as used herein aminoacid or nucleic acid “homology” is equivalent to amino acid or nucleicacid “identity”). The percent homology between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100).

[0066] The determination of percent homology between two sequences canbe accomplished using a mathematical algorithim. A preferred,non-limiting example of a mathematical algorithim utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed performed with the NBLAST program, score=100, wordlength=12 toobtain nucleotide sequences homologous to mACHR-6 nucleic acid moleculesof the invention. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to mACHR-6 protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Research25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. Another preferred,non-limiting example of a mathematical algorithim utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

[0067] An isolated nucleic acid molecule encoding an mACHR-6 polypeptidehomologous to the polypeptide of SEQ ID NO:2, 5, or 32 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 4, or 31, respectively,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded polypeptide. Mutations can be introducedinto SEQ ID NO:1, 4, or 31 by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues., A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), non-polar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in mACHR-6is preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of an mACHR-6 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for an mACHR-6 activity described herein to identify mutantsthat retain mACHR-6 activity. Following mutagenesis of SEQ ID NO:1, 4,or 31, the encoded polypeptide can be expressed recombinantly (e.g., asdescribed in Examples 3 and 4) and the activity of the polypeptide canbe determined using, for example, assays described herein.

[0068] In addition to the nucleic acid molecules encoding mACHR-6polypeptides described above, another aspect of the invention pertainsto isolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a polypeptide, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire mACHR-6 coding strand, or to onlya portion thereof. In one embodiment, an antisense nucleic acid moleculeis antisense to a “coding region” of the coding strand of a nucleotidesequence encoding mACHR-6.

[0069] The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acidresidues, e.g., the entire coding region of SEQ ID NO:1 comprisesnucleotides 291 to 1628 (shown separately as SEQ ID NO:3) and the codingregion of SEQ ID NO:4 comprises nucleotides 778 to 2112 (shownseparately as SEQ ID NO:6). In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding mACHR-6. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

[0070] Given the coding strand sequences encoding mACHR-6 disclosedherein (e.g., SEQ ID NOs:1, 4, and 31), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of mACHR-6 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of mACHR-6 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of mACHR-6 mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil,5-methoxyuracil-2-methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0071] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding anmACHR-6 polypeptide to thereby inhibit expression of the polypeptide,e.g., by inhibiting transcription and/or translation. The hybridizationcan be by conventional nucleotide complementarity to form a stableduplex, or, for example, in the case of an antisense nucleic acidmolecule which binds to DNA duplexes, through specific interactions inthe major groove of the double helix. An example of a route ofadministration of an antisense nucleic acid molecule of the inventionincludes direct injection at a tissue site. Alternatively, an antisensenucleic acid molecule can be modified to target selected cells and thenadministered systemically. For example, for systemic administration, anantisense molecule can be modified such that it specifically binds to areceptor or an antigen expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecule to a peptide or an antibodywhich binds to a cell surface receptor or antigen. The antisense nucleicacid molecule can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0072] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0073] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave mACHR-6 mRNA transcripts to thereby inhibittranslation of mACHR-6 mRNA. A ribozyme having specificity for anmACHR-6-encoding nucleic acid can be designed based upon the nucleotidesequence of an mACHR-6 cDNA disclosed herein (i.e., SEQ ID NO:1, 4, or31). For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in anmACHR-6-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, mACHR-6 mRNA canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel, D. andSzostak, J. W. (1993) Science 261:1411-1418.

[0074] Alternatively, mACHR-6 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe mACHR-6 (e.g., the mACHR-6 promoter and/or enhancers) to form triplehelical structures that prevent transcription of the mACHR-6 gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

[0075] II. Recombinant Expression Vectors and Host Cells

[0076] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding mACHR-6 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0077] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce polypeptides or peptides, including fusionpolypeptides or peptides, encoded by nucleic acids as described herein(e.g., mACHR-6 polypeptides, mutant forms of mACHR-6, fusionpolypeptides, and the like).

[0078] The recombinant expression vectors of the invention can bedesigned for expression of mACHR-6 in prokaryotic or eukaryotic cells.For example, mACHR-6 can be expressed in bacterial cells such as E.coli, insect cells (e.g., using baculovirus expression vectors) yeastcells or mammalian cells. Suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0079] Expression of polypeptides in prokaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: 1) to increase expression of recombinant polypeptide; 2) toincrease the solubility of the recombinant polypeptide; and 3) to aid inthe purification of the recombinant polypeptide by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide to enable separation of therecombinant polypeptide from the fusion moiety subsequent topurification of the fusion polypeptide. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant polypeptide. In oneembodiment, the coding sequence of the mACHR-6 is cloned into a pGEXexpression vector to create a vector encoding a fusion polypeptidecomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-mACHR-6. The fusion polypeptide can be purified by affinitychromatography using glutathione-agarose resin. Recombinant mACHR-6unfused to GST can be recovered by cleavage of the fusion polypeptidewith thrombin.

[0080] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0081] One strategy to maximize recombinant polypeptide expression in E.coli is to express the polypeptide in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant polypeptide(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 119-128). Another strategy isto alter the nucleic acid sequence of the nucleic acid to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in E. coli (Wada et al. (1992)Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

[0082] In another embodiment, the mACHR-6 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

[0083] Alternatively, mACHR-6 can be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of polypeptides in cultured insect cells (e.g.,Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

[0084] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0085] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal. (1985) Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0086] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to mACHR-6 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0087] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0088] A host cell can be any prokaryotic or eukaryotic cell. Forexample, mACHR-6 polypeptide can be expressed in bacterial cells such asE. coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0089] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

[0090] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding mACHR-6 or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0091] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) mACHR-6polypeptide. Accordingly, the invention further provides methods forproducing mACHR-6 polypeptide using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding mACHR-6has been introduced) in a suitable medium until mACHR-6 is produced. Inanother embodiment, the method further comprises isolating mACHR-6 fromthe medium or the host cell.

[0092] The host cells of the invention can also be used to producenon-human transgenic animals. The non-human transgenic animals can beused in screening assays designed to identify agents or compounds, e.g.,drugs, pharmaceuticals, etc., which are capable of amelioratingdetrimental symptoms of selected disorders such as nervous systemdisorders, smooth muscle related disorders, cardiac muscle relateddisorders and gland related disorders. For example, in one embodiment, ahost cell of the invention is a fertilized oocyte or an embryonic stemcell into which mACHR-6-coding sequences have been introduced. Such hostcells can then be used to create non-human transgenic animals in whichexogenous mACHR-6 sequences have been introduced into their genome orhomologous recombinant animals in which endogenous mACHR-6 sequenceshave been altered. Such animals are useful for studying the functionand/or activity of mACHR-6 and for identifying and/or evaluatingmodulators of mACHR-6 activity. As used herein, a “transgenic animal” isa non-human animal, preferably a mammal, more preferably a rodent suchas a rat or mouse, in which one or more of the cells of the animalinclude a transgene. Other examples of transgenic animals includenon-human primates, sheep, dogs, cows, goats, chickens, amphibians, andthe like. A transgene is exogenous DNA which is integrated into thegenome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous mACHR-6 gene has been alteredby homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

[0093] A transgenic animal of the invention can be created byintroducing mACHR-6-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The human mACHR-6 cDNA sequence of SEQ ID NO:1 can be introduced as atransgene into the genome of a non-human animal. Furthermore, the ratmACHR-6 cDNA sequence of SEQ ID NO:4 can be introduced as a transgeneinto the genome of a non-rat animal. Moreover, a non-human homologue ofthe human mACHR-6 gene, such as a mouse mACHR-6 gene, can be isolatedbased on hybridization to the human or rat mACHR-6 cDNA (describedfurther in subsection I above) and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to themACHR-6 transgene to direct expression of mACHR-6 polypeptide toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the mACHR-6 transgene in its genome and/or expression ofmACHR-6 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingmACHR-6 can further be bred to other transgenic animals carrying othertransgenes.

[0094] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of an mACHR-6 gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the mACHR-6 gene. The mACHR-6 gene can be ahuman gene (e.g., from a human genomic clone isolated from a humangenomic library screened with the cDNA of SEQ ID NO:1), but morepreferably, is a rat mACHR-6 gene of SEQ ID NO:4 or 31, or anothernon-human homologue of a human mACHR-6 gene. For example, a mousemACHR-6 gene can be isolated from a mouse genomic DNA library using themACHR-6 cDNA of SEQ ID NO:1, 4, or 31 as a probe. The mouse mACHR-6 genethen can be used to construct a homologous recombination vector suitablefor altering an endogenous mACHR-6 gene in the mouse genome. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous mACHR-6 gene is functionally disrupted(i.e., no longer encodes a functional polypeptide; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous mACHR-6 gene ismutated or otherwise altered but still encodes functional polypeptide(e.g., the upstream regulatory region can be altered to thereby alterthe expression of the endogenous mACHR-6 polypeptide). In the homologousrecombination vector, the altered portion of the mACHR-6 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the mACHR-6 gene toallow for homologous recombination to occur between the exogenousmACHR-6 gene carried by the vector and an endogenous mACHR-6 gene in anembryonic stem cell. The additional flanking mACHR-6 nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see for example, Thomas,K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced mACHR-6 gene has homologously recombined with theendogenous mACHR-6 gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells are then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley, A. (1991) Current Opinion inBiotechnology 2:823-829 and in PCT International Publication Nos. WO90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

[0095] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected polypeptide arerequired. Such animals can be provided through the construction of“double” transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected polypeptide and the othercontaining a transgene encoding a recombinase.

[0096] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0097] III. Isolated mACHR-6 polypeptides and Anti-mACHR-6 Antibodies

[0098] Another aspect of the invention pertains to isolated mACHR-6polypeptides, and biologically active portions thereof, as well aspeptide fragments suitable for use as immunogens to raise anti-mACHR-6antibodies. An “isolated” or “purified” polypeptide or biologicallyactive portion thereof is substantially free of cellular material whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of mACHR-6 polypeptide inwhich the polypeptide is separated from cellular components of the cellsin which it is naturally or recombinantly produced. In one embodiment,the language “substantially free of cellular material” includespreparations of mACHR-6 polypeptide having less than about 30% (by dryweight) of non-mACHR-6 polypeptide (also referred to herein as a“contaminating polypeptide”), more preferably less than about 20% ofnon-mACHR-6 polypeptide, still more preferably less than about 10% ofnon-mACHR-6 polypeptide, and most preferably less than about 5%non-mACHR-6 polypeptide. When the mACHR-6 polypeptide or biologicallyactive portion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the polypeptidepreparation. The language “substantially free of chemical precursors orother chemicals” includes preparations of mACHR-6 polypeptide in whichthe polypeptide is separated from chemical precursors or other chemicalswhich are involved in the synthesis of the polypeptide. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of mACHR-6 polypeptide havingless than about 30% (by dry weight) of chemical precursors ornon-mACHR-6 chemicals, more preferably less than about 20% chemicalprecursors or non-mACHR-6 chemicals, still more preferably less thanabout 10% chemical precursors or non-mACHR-6 chemicals, and mostpreferably less than about 5% chemical precursors or non-mACHR-6chemicals. In preferred embodiments, isolated polypeptides orbiologically active portions thereof lack contaminating polypeptidesfrom the same animal from which the mACHR-6 polypeptide is derived.Typically, such polypeptides are produced by recombinant expression of,for example, a human mACHR-6 polypeptide in a non-human cell.

[0099] An isolated mACHR-6 polypeptide or a portion thereof of theinvention can modulate an acetylcholine response in an acetylcholineresponsive cell or be a naturally occurring, non-functional allelicvariant of an mACHR-6 polypeptide. In preferred embodiments, thepolypeptide or portion thereof comprises an amino acid sequence which issufficiently homologous to an amino acid sequence of SEQ ID NO:2, 5, or32 such that the polypeptide or portion thereof maintains the ability tomodulate an acetylcholine response in an acetylcholine responsive cell.The portion of the polypeptide is preferably a biologically activeportion as described herein. In another preferred embodiment, the humanmACHR-6 polypeptide (i.e., amino acid residues 1-398 of SEQ ID NO:2) orthe rat mACHR-6 polypeptide (i.e., amino acid residues 1-445 of SEQ IDNO:5 or amino acid residues 1-401 of SEQ ID NO:32) has an amino acidsequence shown in SEQ ID NO:2, 5, or 32, respectively, or an amino acidsequence which is encoded by the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC® as Accession Number ______. In yetanother preferred embodiment, the mACHR-6 polypeptide has an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, to the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC® as AccessionNumber ______. In still another preferred embodiment, the mACHR-6polypeptide has an amino acid sequence which is encoded by a nucleotidesequence that is at least about 30-35%, preferably at least about 4045%,more preferably at least about 50-55%, even more preferably at leastabout 60-65%, yet more preferably at least about 70-75%, still morepreferably at least about 80-85%, and most preferably at least about90-95% or more homologous to the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC® as Accession Number ______. Thepreferred mACHR-6 polypeptides of the present invention also preferablypossess at least one of the mACHR-6 activities described herein. Forexample, a preferred mACHR-6 polypeptide of the present inventionincludes an amino acid sequence encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, to thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______ and which can modulate an acetylcholineresponse in an acetylcholine responsive cell.

[0100] In other embodiments, the mACHR-6 polypeptide is substantiallyhomologous to the amino acid sequence of SEQ ID NO:2, 5, or 32 andretains the functional activity of the polypeptide of SEQ ID NO:2, 5, or32 yet differs in amino acid sequence due to natural allelic variationor mutagenesis, as described in detail in subsection I above.Accordingly, in another embodiment, the mACHR-6 polypeptide is apolypeptide which comprises an amino acid sequence which is at leastabout 30-35%, preferably at least about 40-45%, more preferably at leastabout 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more homologous tothe amino acid sequence of SEQ ID NO:2, 5, or 32 and which has at leastone of the mACHR-6 activities described herein. In still otherembodiments, the invention pertains to a full length human polypeptidewhich is substantially homologous to the entire amino acid sequence ofSEQ ID NO:2, 5, or 32. In still another embodiment, the inventionpertains to nonfunctional, naturally occurring allelic variants of themACHR-6 polypeptides described herein. Such allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion or premature truncation of the amino acid sequence of SEQ IDNO:2, 5, or 32.

[0101] Biologically active portions of the mACHR-6 polypeptide includepeptides comprising amino acid sequences derived from the amino acidsequence of the mACHR-6 polypeptide, e.g., the amino acid sequence shownin SEQ ID NO:2, 5, or 32 or the amino acid sequence of a polypeptidehomologous to the mACHR-6 polypeptide, which include less amino acidsthan the full length mACHR-6 polypeptide or the full length polypeptidewhich is homologous to the mACHR-6 polypeptide, and exhibit at least oneactivity of the mACHR-6 polypeptide. Typically, biologically activeportions (peptides, e.g., peptides which are, for example, 5, 10, 15,20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length)comprise a domain or motif, e.g., a transmembrane domain, with at leastone activity of the mACHR-6 polypeptide. Preferably, the domain is atransmembrane domain derived from a human and is at least about 75-80%,preferably at least about 80-85%, more preferably at least about 85-90%,and most preferably at least about 90-95% or more homologous to SEQ IDNO:7, 8, 9, 10, 11, 12, or 13 or to the corresponding rat sequences. Ina preferred embodiment, the biologically active portion of thepolypeptide which includes the transmembrane domain can modulate theactivity of a G protein in a cell and/or modulate an acetylcholineresponse in a cell, e.g., an acetylcholine responsive cell, e.g., abrain cell, to thereby beneficially affect the acetylcholine responsivecell. In a preferred embodiment, the biologically active portioncomprises a transmembrane domain of mACHR-6 as represented by amino acidresidues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8), 109-130 (SEQ IDNO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11), 360-380 (SEQ IDNO:12), and 396416 (SEQ ID NO:13), or the corresponding rat sequencesshown in SEQ ID NOs:14-20 amd 34-39. Moreover, other biologically activeportions, in which other regions of the polypeptide are deleted, can beprepared by recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of the mACHR-6 polypeptide include one or more selecteddomains/motifs or portions thereof having biological activity.

[0102] mACHR-6 polypeptides are preferably produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding thepolypeptide is cloned into an expression vector (as described above),the expression vector is introduced into a host cell (as describedabove) and the mACHR-6 polypeptide is expressed in the host cell. ThemACHR-6 polypeptide can then be isolated from the cells by anappropriate purification scheme using standard polypeptide purificationtechniques. Alternative to recombinant expression, an mACHR-6polypeptide, protein, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native mACHR-6polypeptide can be isolated from cells (e.g., hippocampal cells,substantia nigra cells, or parotid gland cells), for example using ananti-mACHR-6 antibody (described further below).

[0103] The invention also provides mACHR-6 chimeric or fusionpolypeptides. As used herein, an mACHR-6 “chimeric polypeptide” or“fusion polypeptide” comprises an mACHR-6 polypeptide operatively linkedto a non-mACHR-6 polypeptide. An “mACHR-6 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to mACHR-6,whereas a “non-mACHR-6 polypeptide” refers to a heterologous polypeptidehaving an amino acid sequence corresponding to a polypeptide which isnot substantially homologous to the mACHR-6 polypeptide, e.g., apolypeptide which is different from the mACHR-6 polypeptide and which isderived from the same or a different organism. Within the fusionpolypeptide, the term “operatively linked” is intended to indicate thatthe mACHR-6 polypeptide and the non-mACHR-6 polypeptide are fusedin-frame to each other. The non-mACHR-6 polypeptide can be fused to theN-terminus or C-terminus of the mACHR-6 polypeptide. For example, in oneembodiment the fusion polypeptide is a GST-mACHR-6 fusion polypeptide inwhich the mACHR-6 sequences are fused to the C-terminus of the GSTsequences. Other types of fusion polypeptides include, but are notlimited to, enzymatic fusion polypeptides, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly Hisfusions and Ig fusions. Such fusion polypeptides, particularly poly Hisfusions, can facilitate the purification of recombinant mACHR-6. Inanother embodiment, the fusion polypeptide is an mACHR-6 polypeptidecontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofmACHR-6 can be increased through use of a heterologous signal sequence.

[0104] Preferably, an mACHR-6 chimeric or fusion polypeptide of theinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). An mACHR-6-encoding nucleic acid can be cloned into suchan expression vector such that the fusion moiety is linked in-frame tothe mACHR-6 polypeptide.

[0105] The present invention also pertains to homologues of the mACHR-6polypeptides which function as either an mACHR-6 agonist (mimetic) or anmACHR-6 antagonist. In a preferred embodiment, the mACHR-6 agonists andantagonists stimulate or inhibit, respectively, a subset of thebiological activities of the naturally occurring form of the mACHR-6polypeptide. Thus, specific biological effects can be elicited bytreatment with a homologue of limited function. In one embodiment,treatment of a subject with a homologue having a subset of thebiological activities of the naturally occurring form of the polypeptidehas fewer side effects in a subject relative to treatment with thenaturally occurring form of the mACHR-6 polypeptide.

[0106] Homologues of the mACHR-6 polypeptide can be generatedby-mutagenesis, e.g., discrete point mutation or truncation of themACHR-6 polypeptide. As used herein, the term “homologue” refers to avariant form of the mACHR-6 polypeptide which acts as an agonist orantagonist of the activity of the mACHR-6 polypeptide. An agonist of themACHR-6 polypeptide can retain substantially the same, or a subset, ofthe biological activities of the mACHR-6 polypeptide. An antagonist ofthe mACHR-6 polypeptide can inhibit one or more of the activities of thenaturally occurring form of the mACHR-6 polypeptide, by, for example,competitively binding to a downstream or upstream member of the mACHR-6cascade which includes the mACHR-6 polypeptide. Thus, the mammalianmACHR-6 polypeptide and homologues thereof of the present invention canbe either positive or negative regulators of acetylcholine responses inacetylcholine responsive cells.

[0107] In an alternative embodiment, homologues of the mACHR-6polypeptide can be identified by screening combinatorial libraries ofmutants, e.g., truncation mutants, of the mACHR-6 polypeptide formACHR-6 polypeptide agonist or antagonist activity. In one embodiment, avariegated library of mACHR-6 variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of mACHR-6 variants can be producedby, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential mACHR-6 sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion polypeptides (e.g., forphage display) containing the set of mACHR-6 sequences therein. Thereare a variety of methods which can be used to produce libraries ofpotential mACHR-6 homologues from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential mACHR-6 sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477).

[0108] In addition, libraries of fragments of the mACHR-6 polypeptidecoding can be used to generate a variegated population of mACHR-6fragments for screening and subsequent selection of homologues of anmACHR-6 polypeptide. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofan mACHR-6 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal, C-terminal and internal fragments of various sizesof the mACHR-6 polypeptide.

[0109] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of mACHR-6homologues. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recrusive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify mACHR-6 homologues (Arkin and Yourvan(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

[0110] In one embodiment, cell based assays can be exploited to analyzea variegated mACHR-6 library. For example, a library of expressionvectors can be transfected into a cell line ordinarily responsive toacetylcholine. The transfected cells are then contacted withacetylcholine and the effect of the mACHR-6 mutant on signaling byacetylcholine can be detected, e.g., by measuring intracellular calciumconcentration. Plasmid DNA can then be recovered from the cells whichscore for inhibition, or alternatively, potentiation of acetylcholineinduction, and the individual clones further characterized.

[0111] An isolated mACHR-6 polypeptide, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindmACHR-6 using standard techniques for polyclonal and monoclonal antibodypreparation. The full-length mACHR-6 polypeptide can be used or,alternatively, the invention provides antigenic peptide fragments ofmACHR-6 for use as immunogens. The antigenic peptide of mACHR-6comprises at least 8 amino acid residues of the amino acid sequenceshown in SEQ ID NO:2, 5, or 32 and encompasses an epitope of mACHR-6such that an antibody raised against the peptide forms a specific immunecomplex with mACHR-6. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of mACHR-6 that arelocated on the surface of the polypeptide, e.g., hydrophilic regions.

[0112] An mACHR-6 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed mACHR-6 polypeptide or achemically synthesized mACHR-6 peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic mACHR-6 preparation induces a polyclonalanti-mACHR-6 antibody response.

[0113] Accordingly, another aspect of the invention pertains toanti-mACHR-6 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as mACHR-6. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bindmACHR-6. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of mACHR-6. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular mACHR-6 polypeptide with which itimmunoreacts.

[0114] Polyclonal anti-mACHR-6 antibodies can be prepared as describedabove by immunizing a suitable subject with an mACHR-6 immunogen. Theanti-mACHR-6 antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized mACHR-6. If desired, theantibody molecules directed against mACHR-6 can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-mACHR-6antibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:53946; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982)Int. J. Cancer 29:269-75), the more recent human B cell hybridomatechnique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridomatechnique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387402; M. L. Gefter et al. (1977) SomaticCell Genet. 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an mACHR-6 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds mACHR-6.

[0115] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-mACHR-6 monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC®.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindmACHR-6, e.g., using a standard ELISA assay.

[0116] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-mACHR-6 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with mACHR-6 to therebyisolate immunoglobulin library members that bind mACHR-6. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT International Publication No. WO92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature(1990) 348:552-554.

[0117] Additionally, recombinant anti-mACHR-6 antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. PCT International Application No. PCT/US86/02269; Akira,et al. European Patent Application 184,187; Taniguchi, M., EuropeanPatent Application 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S.L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214;Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

[0118] An anti-mACHR-6 antibody (e.g., monoclonal antibody) can be usedto isolate mACHR-6 by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-mACHR-6 antibody canfacilitate the purification of natural mACHR-6 from cells and ofrecombinantly produced mACHR-6 expressed in host cells. Moreover, ananti-mACHR-6 antibody can be used to detect mACHR-6 polypeptide (e.g.,in a cellular lysate or cell supernatant) in order to evaluate theabundance and pattern of expression of the mACHR-6 polypeptide or afragment of an mACHR-6 polypeptide. The detection of circulatingfragments of an mACHR-6 polypeptide can be used to identify mACHR-6turnover in a subject. Anti-mACHR-6 antibodies can be useddiagnostically to monitor polypeptide levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0119] IV. Pharmaceutical Compositions

[0120] The mACHR-6 nucleic acid molecules, mACHR-6 polypeptides(particularly fragments of mACHR-6), mACHR-6 modulators, andanti-mACHR-6 antibodies (also referred to herein as “active compounds”)of the invention can be incorporated into pharmaceutical compositionssuitable for administration to a subject, e.g., a human. Suchcompositions typically comprise the nucleic acid molecule, polypeptide,modulator, or antibody and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions.

[0121] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0122] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0123] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., an mACHR-6 polypeptide or anti-mACHR-6 antibody)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0124] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0125] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0126] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0127] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0128] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0129] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0130] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0131] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0132] V. Uses and Methods of the Invention

[0133] The nucleic acid molecules, polypeptides, polypeptide homologues,modulators, and antibodies described herein can be used in one or moreof the following methods: a) drug screening assays; b) diagnostic assaysparticularly in disease identification, allelic screening andpharmocogenetic testing; c) methods of treatment; d) pharmacogenomics;and e) monitoring of effects during clinical trials. An mACHR-6polypeptide of the invention has one or more of the activities describedherein and can thus be used to, for example, modulate an acetylcholineresponse in an acetylcholine responsive cell, for example by binding toacetylcholine or an mACHR-6 binding partner making it unavailable forbinding to the naturally present mACHR-6 polypeptide. The isolatednucleic acid molecules of the invention can be used to express mACHR-6polypeptide (e.g., via a recombinant expression vector in a host cell orin gene therapy applications), to detect mACHR-6 mRNA (e.g., in abiological sample) or a naturally occurring or recombinantly generatedgenetic mutation in an mACHR-6 gene, and to modulate mACHR-6 activity,as described further below. In addition, the mACHR-6 polypeptides can beused to screen drugs or compounds which modulate mACHR-6 polypeptideactivity as well as to treat disorders characterized by insufficientproduction of mACHR-6 polypeptide or production of mACHR-6 polypeptideforms which have decreased activity compared to wild type mACHR-6.Moreover, the anti-mACHR-6 antibodies of the invention can be used todetect and isolate an mACHR-6 polypeptide, particularly fragments ofmACHR-6 present in a biological sample, and to modulate mACHR-6polypeptide activity.

[0134] a. Drug Screening Assays:

[0135] The invention provides methods for identifying compounds oragents which can be used to treat disorders characterized by (orassociated with) aberrant or abnormal mACHR-6 nucleic acid expressionand/or mACHR-6 polypeptide activity. These methods are also referred toherein as drug screening assays and typically include the step ofscreening a candidate/test compound or agent to be an agonist orantagonist of mACHR-6, and specifically for the ability to interact with(e.g., bind to) an mACHR-6 polypeptide, to modulate the interaction ofan mACHR-6 polypeptide and a target molecule, and/or to modulate mACHR-6nucleic acid expression and/or mACHR-6 polypeptide activity.Candidate/test compounds or agents which have one or more of theseabilities can be used as drugs to treat disorders characterized byaberrant or abnormal mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity. Candidate/test compounds include, for example, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam, K. S. et al.(1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86)and combinatorial chemistry-derived molecular libraries made of D-and/or L-configuration amino acids; 2) phosphopeptides (e.g., members ofrandom and partially degenerate, directed phosphopeptide libraries, see,e.g., Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

[0136] In one embodiment, the invention provides assays for screeningcandidate/test compounds which interact with (e.g., bind to) mACHR-6polypeptide. Typically, the assays are recombinant cell based orcell-free assays which include the steps of combining an mACHR-6polypeptide or a bioactive fragment thereof, and a candidate/testcompound, e.g., under conditions which allow for interaction of (e.g.,binding of) the candidate/test compound to the mACHR-6 polypeptide orfragment thereof to form a complex, and detecting the formation of acomplex, in which the ability of the candidate compound to interact with(e.g., bind to) the mACHR-6 polypeptide or fragment thereof is indicatedby the presence of the candidate compound in the complex. Formation ofcomplexes between the mACHR-6 polypeptide and the candidate compound canbe quantitated, for example, using standard immunoassays.

[0137] In another embodiment, the invention provides screening assays toidentify candidate/test compounds which modulate (e.g., stimulate orinhibit) the interaction (and most likely mACHR-6 activity as well)between an mACHR-6 polypeptide and a molecule (target molecule) withwhich the mACHR-6 polypeptide normally interacts. Examples of suchtarget molecules include polypeptides in the same signaling path as themACHR-6 polypeptide, e.g., polypeptides which may function upstream(including both stimulators and inhibitors of activity) or downstream ofthe mACHR-6 polypeptide in, for example, a cognitive function signalingpathway or in a pathway involving mACHR-6 activity, e.g., a G protein orother interactor involved in phosphatidylinositol turnover and/orphospholipase C activation. Typically, the assays are recombinant cellbased or cell-free assays which include the steps of combining a cellexpressing an mACHR-6 polypeptide, or a bioactive fragment thereof, anmACHR-6 target molecule (e.g., an mACHR-6 ligand) and a candidate/testcompound, e.g., under conditions wherein but for the presence of thecandidate compound, the mACHR-6 polypeptide or biologically activeportion thereof interacts with (e.g., binds to) the target molecule, anddetecting the formation of a complex which includes the mACHR-6polypeptide and the target molecule or detecting theinteraction/reaction of the mACHR-6 polypeptide and the target molecule.Detection of complex formation can include direct quantitation of thecomplex by, for example, measuring inductive effects of the mACHR-6polypeptide. A statistically significant change, such as a decrease, inthe interaction of the mACHR-6 and target molecule (e.g., in theformation of a complex between the mACHR-6 and the target molecule) inthe presence of a candidate compound (relative to what is detected inthe absence of the candidate compound) is indicative of a modulation(e.g., stimulation or inhibition) of the interaction between the mACHR-6polypeptide and the target molecule. Modulation of the formation ofcomplexes between the mACHR-6 polypeptide and the target molecule can bequantitated using, for example, an immunoassay.

[0138] To perform cell free drug screening assays, it is desirable toimmobilize either mACHR-6 or its target molecule to facilitateseparation of complexes from uncomplexed forms of one or both of thepolypeptides, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of mACHR-6 to a target molecule, in thepresence and absence of a candidate compound, can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtitre plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion polypeptide can be provided which adds a domainthat allows the polypeptide to be bound to a matrix. For example,glutathione-S-transferase/mACHR-6 fusion polypeptides can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofmACHR-6-binding polypeptide found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques.

[0139] Other techniques for immobilizing polypeptides on matrices canalso be used in the drug screening assays of the invention. For example,either mACHR-6 or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated mACHR-6 moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withmACHR-6 but which do not interfere with binding of the polypeptide toits target molecule can be derivatized to the wells of the plate, andmACHR-6 trapped in the wells by antibody conjugation. As describedabove, preparations of an mACHR-6-binding polypeptide and a candidatecompound are incubated in the mACHR-6-presenting wells of the plate, andthe amount of complex trapped in the well can be quantitated. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with the mACHR-6 target molecule, or which arereactive with mACHR-6 polypeptide and compete with the target molecule;as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0140] In yet another embodiment, the invention provides a method foridentifying a compound (e.g., a screening assay) capable of use in thetreatment of a disorder characterized by (or associated with) aberrantor abnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity. This method typically includes the step of assaying theability of the compound or agent to modulate the expression of themACHR-6 nucleic acid or the activity of the mACHR-6 polypeptide therebyidentifying a compound for treating a disorder characterized by aberrantor abnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity. Disorders characterized by aberrant or abnormal mACHR-6nucleic acid expression or mACHR-6 polypeptide activity are describedherein. Methods for assaying the ability of the compound or agent tomodulate the expression of the mACHR-6 nucleic acid or activity of themACHR-6 polypeptide are typically cell-based assays. For example, cellswhich are sensitive to ligands which transduce signals via a pathwayinvolving mACHR-6 can be induced to overexpress an mACHR-6 polypeptidein the presence and absence of a candidate compound. Candidate compoundswhich produce a statistically significant change in mACHR-6-dependentresponses (either stimulation or inhibition) can be identified. In oneembodiment, expression of the mACHR-6 nucleic acid or activity of anmACHR-6 polypeptide is modulated in cells and the effects of candidatecompounds on the readout of interest (such as phosphatidylinositolturnover) are measured. For example, the expression of genes which areup- or down-regulated in response to an mACHR-6-dependent signal cascadecan be assayed. In preferred embodiments, the regulatory regions of suchgenes, e.g., the 5′ flanking promoter and enhancer regions, are operablylinked to a detectable marker (such as luciferase) which encodes a geneproduct that can be readily detected. Phosphorylation of mACHR-6 ormACHR-6 target molecules can also be measured, for example, byimmunoblotting.

[0141] Alternatively, modulators of mACHR-6 expression (e.g., compoundswhich can be used to treat a disorder characterized by aberrant orabnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity) can be identified in a method wherein a cell is contacted witha candidate compound and the expression of mACHR-6 mRNA or polypeptidein the cell is determined. The level of expression of mACHR-6 mRNA orpolypeptide in the presence of the candidate compound is compared to thelevel of expression of mACHR-6 mRNA or polypeptide in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of mACHR-6 nucleic acid expression based on this comparisonand be used to treat a disorder characterized by aberrant mACHR-6nucleic acid expression. For example, when expression of mACHR-6 mRNA orpolypeptide is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of mACHR-6 nucleic acidexpression. Alternatively, when mACHR-6 nucleic acid expression is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of mACHR-6 nucleic acid expression. The level of mACHR-6nucleic acid expression in the cells can be determined by methodsdescribed herein for detecting mACHR-6 mRNA or polypeptide.

[0142] In yet another aspect of the invention, the mACHR-6 polypeptides,or fragments thereof, can be used as “bait proteins” in a two-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO 94/10300), to identify other proteins, whichbind to or interact with mACHR-6 (“mACHR-6-binding proteins” or“mACHR-6-bp”) and modulate mACHR-6 polypeptide activity. SuchmACHR-6-binding proteins are also likely to be involved in thepropagation of signals by the mACHR-6 polypeptides as, for example,upstream or downstream elements of the mACHR-6 pathway.

[0143] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Bartel, P. et al. “Using the Two-Hybrid System toDetect Protein-Protein Interactions” in Cellular Interactions inDevelopment: A Practical Approach, Hartley, D. A. ed. (Oxford UniversityPress, Oxford, 1993) pp. 153-179. Briefly, the assay utilizes twodifferent DNA constructs. In one construct, the gene that codes formACHR-6 is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming anmACHR-6-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with mACHR-6.

[0144] Modulators of mACHR-6 polypeptide activity and/or mACHR-6 nucleicacid expression identified according to these drug screening assays canbe used to treat, for example, nervous system disorders, smooth musclerelated disorders, cardiac muscle related disorders, and gland relateddisorders. These methods of treatment include the steps of administeringthe modulators of mACHR-6 polypeptide activity and/or nucleic acidexpression, e.g., in a pharmaceutical composition as described insubsection IV above, to a subject in need of such treatment, e.g., asubject with a disorder described herein.

[0145] b. Diagnostic Assays:

[0146] The invention further provides a method for detecting thepresence of mACHR-6, or fragment thereof, in a biological sample. Themethod involves contacting the biological sample with a compound or anagent capable of detecting mACHR-6 polypeptide or mRNA such that thepresence of mACHR-6 is detected in the biological sample. A preferredagent for detecting mACHR-6 mRNA is a labeled or labelable nucleic acidprobe capable of hybridizing to mACHR-6 mRNA. The nucleic acid probe canbe, for example, the full-length mACHR-6 cDNA of SEQ ID NO:1, 4, or 31,or a portion thereof, such as an oligonucleotide of at least 15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to mACHR-6 mRNA. A preferred agentfor detecting mACHR-6 polypeptide is a labeled or labelable antibodycapable of binding to mACHR-6 polypeptide. Antibodies can be polyclonal,or more preferably, monoclonal. An intact antibody, or a fragmentthereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled orlabelable”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect mACHR-6 mRNA or polypeptide in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of mACHR-6 mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of mACHR-6 polypeptideinclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, mACHR-6polypeptide can be detected in vivo in a subject by introducing into thesubject a labeled anti-mACHR-6 antibody. For example, the antibody canbe labeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. Particularlyuseful are methods which detect the allelic variant of mACHR-6 expressedin a subject and methods which detect fragments of an mACHR-6polypeptide in a sample.

[0147] The invention also encompasses kits for detecting the presence ofmACHR-6 in a biological sample. For example, the kit can comprise alabeled or labelable compound or agent capable of detecting mACHR-6polypeptide or mRNA in a biological sample; means for determining theamount of mACHR-6 in the sample; and means for comparing the amount ofmACHR-6 in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect mACHR-6 mRNA or polypeptide.

[0148] The methods of the invention can also be used to detect naturallyoccurring genetic mutations in an mACHR-6 gene, thereby determining if asubject with the mutated gene is at risk for a disorder characterized byaberrant or abnormal mACHR-6 nucleic acid expression or mACHR-6polypeptide activity as described herein. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic mutation characterized by at least oneof an alteration affecting the integrity of a gene encoding an mACHR-6polypeptide, or the misexpression of the mACHR-6 gene. For example, suchgenetic mutations can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from an mACHR-6gene; 2) an addition of one or more nucleotides to an mACHR-6 gene; 3) asubstitution of one or more nucleotides of an mACHR-6 gene, 4) achromosomal rearrangement of an mACHR-6 gene; 5) an alteration in thelevel of a messenger RNA transcript of an mACHR-6 gene, 6) aberrantmodification of an mACHR-6 gene, such as of the methylation pattern ofthe genomic DNA, 7) the presence of a non-wild type splicing pattern ofa messenger RNA transcript of an mACHR-6 gene, 8) a non-wild type levelof an mACHR-6-polypeptide, 9) allelic loss of an mACHR-6 gene, and 10)inappropriate post-translational modification of an mACHR-6-polypeptide.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting mutations in an mACHR-6 gene.

[0149] In certain embodiments, detection of the mutation involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) PNAS 91:360-364), the latter of which can be particularly usefulfor detecting point mutations in the mACHR-6-gene (see Abravaya et al.(1995) Nucleic Acids Res 0.23:675-682). This method can include thesteps of collecting a sample of cells from a patient, isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to an mACHR-6 gene under conditions such thathybridization and amplification of the mACHR-6-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample.

[0150] In an alternative embodiment, mutations in an mACHR-6 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0151] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the mACHR-6gene and detect mutations by comparing the sequence of the samplemACHR-6 with the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463).A variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0152] Other methods for detecting mutations in the mACHR-6 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985)Science 230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al.(1992) Meth. Enzymol. 217:286-295), electrophoretic mobility of mutantand wild type nucleic acid is compared (Orita et al. (1989) PNAS86:2766; Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79), and movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (Myers et al (1985) Nature313:495). Examples of other techniques for detecting point mutationsinclude, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

[0153] c. Methods of Treatment

[0154] Another aspect of the invention pertains to methods for treatinga subject, e.g., a human, having a disease or disorder characterized by(or associated with) aberrant or abnormal mACHR-6 nucleic acidexpression and/or mACHR-6 polypeptide activity. These methods includethe step of administering an mACHR-6 modulator (agonist or antagonist)to the subject such that treatment occurs. The language “aberrant orabnormal mACHR-6 expression” refers to expression of a non-wild-typemACHR-6 polypeptide or a non-wild-type level of expression of an mACHR-6polypeptide. Aberrant or abnormal mACHR-6 activity refers to aneon-wild-type mACHR-6 activity or a non-wild-type level of mACHR-6activity. As the mACHR-6 polypeptide is involved in a pathway involvingmodulation of neurotransmitter, e.g., acetylcholine, release; modulationof smooth muscle contraction; modulation of cardiac muscle contraction;and modulation of gland, e.g., exocrine gland function, aberrant orabnormal mACHR-6 activity or expression interferes with the normalneurotransmitter, e.g., acetylcholine, release; normal smooth muscle;and cardiac muscle contraction; and normal gland, e.g., exocrine glandfunction. Non-limiting examples of disorders or diseases characterizedby or associated with abnormal or aberrant mACHR-6 activity orexpression include nervous system related disorders, e.g., centralnervous system related disorders. Examples of nervous system relateddisorders include cognitive disorders, e.g., memory and learningdisorders, such as amnesia, apraxia, agnosia, amnestic dysnomia,amnestic spatial disorientation, Kluver-Bucy syndrome, Alzheimer'srelated memory loss (Eglen R. M. (1996) Pharmacol. and Toxicol.78(2):59-68; Perry E. K. (1995) Brain and Cognition 28(3):240-58) andlearning disability; disorders affecting consciousness, e.g., visualhallucinations, perceptual disturbances, or delerium associated withLewy body dementia; schitzo-effective disorders (Dean B. (1996) Mol.Psychiatry 1(1):54-8), schizophrenia with mood swings (Bymaster F. P.(1997) J. Clin. Psychiatry 58 (suppl. 10):28 36; Yeomans J. S. (1995)Neuropharmacol. 12(1):3-16; Reimann D. (1994) J. Psychiatric Res.28(3):195-210), depressive illness (primary or secondary); affectivedisorders (Janowsky D. S. (1994) Am. J. Med. Genetics 54(4):335-44);sleep disorders (Kimura F. (1997) J. Neurophysiol. 77(2):709-16), e.g.,REM sleep abnormalities in patients suffering from, for example,depression (Riemann D. (1994) J. Psychosomatic Res. 38 Suppl. 1:15-25;Bourgin P. (1995) Neuroreport 6(3): 532-6), paradoxical sleepabnormalities (Sakai K. (1997) Eur. J. Neuroscience 9(3):415-23),sleep-wakefulness, and body temperature or respiratory depressionabnormalities during sleep (Shuman S. L. (1995) Am. J. Physiol. 269(2 Pt2):R308-17; Mallick B. N. (1997) Brain Res. 750(1-2):311-7). Otherexamples of nervous system related disorders include disorders affectingpain generation mechanisms, e.g., pain related to irritable bowelsyndrome (Mitch C. H. (1997) J. Med. Chem. 40(4):538-46; Shannon H. E.(1997) J. Pharmac. and Exp. Therapeutics 281(2):884-94; Bouaziz H.(1995) Anesthesia and Analgesia 80(6):1140-4; or Guimaraes A. P. (1994)Brain Res. 647(2):220-30) or chest pain; movement disorders (Monassi C.R. (1997) Physiol. and Behav. 62(1):53-9), e.g., Parkinson's diseaserelated movement disorders (Finn M. (1997) Pharmacol. Biochem. &Behavior 57(1-2):243-9; Mayorga A. J. (1997) Pharmacol. Biochem. &Behavior 56(2):273-9); eating disorders, e.g., insulin hypersecretionrelated obesity (Maccario M. (1997) J. Endocrinol. Invest. 20(1):8-12;Premawardhana L. D. (1994) Clin. Endocrinol. 40(5): 617-21); or drinkingdisorders, e.g., diabetic polydipsia (Murzi E. (1997) Brain Res.752(1-2):184-8; Yang X. (1994) Pharmacol. Biochem. & Behavior49(1):1-6). Yet further examples of disorders or diseases characterizedby or associated with abnormal or aberrant mACHR-6 activity orexpression include smooth muscle related disorders such as irritablebowel syndrome, diverticular disease, urinary incontinence, oesophagealachalasia, or chronic obstructive airways disease; heart muscle relateddisorders such as pathologic bradycardia or tachycardia, arrhythmia,flutter or fibrillation; or gland related disorders such as xerostomia,or diabetes mellitus. The terms “treating” or “treatment”, as usedherein, refer to reduction or alleviation of at least one adverse effector symptom of a disorder or disease, e.g., a disorder or diseasecharacterized by or associated with abnormal or aberrant mACHR-6polypeptide activity or mACHR-6 nucleic acid expression.

[0155] As used herein, an mACHR-6 modulator is a molecule which canmodulate mACHR-6 nucleic acid expression and/or mACHR-6 polypeptideactivity. For example, an mACHR-6 modulator can modulate, e.g.,upregulate (activate/agonize) or downregulate (suppress/antagonize),mACHR-6 nucleic acid expression. In another example, an mACHR-6modulator can modulate (e.g., stimulate/agonize or inhibit/antagonize)mACHR-6 polypeptide activity. If it is desirable to treat a disorder ordisease characterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by inhibiting mACHR-6 nucleic acid expression, anmACHR-6 modulator can be an antisense molecule, e.g., a ribozyme, asdescribed herein. Examples of antisense molecules which can be used toinhibit mACHR-6 nucleic acid expression include antisense moleculeswhich are complementary to a portion of the 5′ untranslated region ofSEQ ID NO:1 which also includes the start codon and antisense moleculeswhich are complementary to a portion of the 3′ untranslated region ofSEQ ID NO:1, 4, or 31. An example of an antisense molecule which iscomplementary to a portion of the 5′ untranslated region of SEQ ID NO:1and which also includes the start codon is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 280 to 296of SEQ ID NO:1. This antisense molecule has the following nucleotidesequence: 5′CCTGCGGGGCCATGGAG 3′ (SEQ ID NO:21). An example of anantisense molecule which is complementary to a portion of the 3′untranslated region of SEQ ID NO:1 is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 1629 to 1645of SEQ ID NO:1. This antisense molecule has the following sequence: 5′GTGGCCCACCAGAGCCT 3′ (SEQ ID NO:22). An additional example of anantisense molecule which is complementary to a portion of the 3′untranslated region of SEQ ID NO:1 is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 1650 to 1666of SEQ ID NO:1. This antisense molecule has the following sequence:5′CAGCCACGCCTCTCTCA 3′ (SEQ ID NO:23). An example of an antisensemolecule which is complementary to a portion of the 5′ untranslatedregion of SEQ ID NO:4 and which also includes the start codon, is anucleic acid molecule which includes nucleotides which are complementaryto nucleotides 766 to 783 of SEQ ID NO:4. This antisense molecule hasthe following nucleotide sequence: 5′ GCCTGCTGGGCCATGGAG 3′ (SEQ IDNO:24). An example of an antisense molecule which is complementary to aportion of the 3′ untranslated region of SEQ ID NO:4 is a nucleic acidmolecule which includes nucleotides which are complementary tonucleotides 2113 to 2128 of SEQ ID NO:4. This antisense molecule has thefollowing sequence: 5′ TGAGCAGCTGCCCCAC 3′ (SEQ ID NO:25). An additionalexample of an antisense molecule which is complementary to a portion ofthe 3′ untranslated region of SEQ ID NO:4 is a nucleic acid moleculewhich includes nucleotides which are complementary to nucleotides 2133to 2148 of SEQ ID NO:4. This antisense molecule has the followingsequence: 5′CTGAGGCCAGGCCCTT 3′ (SEQ ID NO:26).

[0156] An mACHR-6 modulator which inhibits mACHR-6 nucleic acidexpression can also be a small molecule or other drug, e.g., a smallmolecule or drug identified using the screening assays described herein,which inhibits mACHR-6 nucleic acid expression. If it is desirable totreat a disease or disorder characterized by (or associated with)aberrant or abnormal (non-wild-type) mACHR-6 nucleic acid expressionand/or mACHR-6 polypeptide activity by stimulating mACHR-6 nucleic acidexpression, an mACHR-6 modulator can be, for example, a nucleic acidmolecule encoding mACHR-6 (e.g., a nucleic acid molecule comprising anucleotide sequence homologous to the nucleotide sequence of SEQ IDNO:1, 4, or 31) or a small molecule or other drug, e.g., a smallmolecule (peptide) or drug identified using the screening assaysdescribed herein, which stimulates mACHR-6 nucleic acid expression.

[0157] Alternatively, if it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by inhibiting mACHR-6 polypeptide activity, anmACHR-6 modulator can be an anti-mACHR-6 antibody or a small molecule orother drug, e.g., a small molecule or drug identified using thescreening assays described herein, which inhibits mACHR-6 polypeptideactivity. If it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by stimulating mACHR-6 polypeptide activity, anmACHR-6 modulator can be an active mACHR-6 polypeptide or portionthereof (e.g., an mACHR-6 polypeptide or portion thereof having an aminoacid sequence which is homologous to the amino acid sequence of SEQ IDNO:2, 5, or 32 or a portion thereof) or a small molecule or other drug,e.g., a small molecule or drug identified using the screening assaysdescribed herein, which stimulates mACHR-6 polypeptide activity.

[0158] Other aspects of the invention pertain to methods for modulatinga cell associated activity. These methods include contacting the cellwith an agent (or a composition which includes an effective amount of anagent) which modulates mACHR-6 polypeptide activity or mACHR-6 nucleicacid expression such that a cell associated activity is altered relativeto a cell associated activity (for example, phosphatidylinositolmetabolism) of the cell in the absence of the agent. As used herein, “acell associated activity” refers to a normal or abnormal activity orfunction of a cell. Examples of cell associated activities includephosphatidylinositol turnover, production or secretion of molecules,such as proteins, contraction, proliferation, migration,differentiation, and cell survival. In a preferred embodiment, the cellis neural cell of the brain, e.g., a hippocampal cell. The term“altered” as used herein refers to a change, e.g., an increase ordecrease, of a cell associated activity particularlyphosphatidylinositol turnover and phospholipase C activation. In oneembodiment, the agent stimulates mACHR-6 polypeptide activity or mACHR-6nucleic acid expression. Examples of such stimulatory agents include anactive mACHR-6 polypeptide, a nucleic acid molecule encoding mACHR-6that has been introduced into the cell, and a modulatory agent whichstimulates mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression and which is identified using the drug screening assaysdescribed herein. In another embodiment, the agent inhibits mACHR-6polypeptide activity or mACHR-6 nucleic acid expression. Examples ofsuch inhibitory agents include an antisense mACHR-6 nucleic acidmolecule, an anti-mACHR-6 antibody, and a modulatory agent whichinhibits mACHR-6 polypeptide activity or mACHR-6 nucleic acid expressionand which is identified using the drug screening assays describedherein. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). In a preferred embodiment, themodulatory methods are performed in vivo, i.e., the cell is presentwithin a subject, e.g., a mammal, e.g., a human, and the subject has adisorder or disease characterized by or associated with abnormal oraberrant mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression.

[0159] A nucleic acid molecule, a polypeptide, an mACHR-6 modulator, acompound etc. used in the methods of treatment can be incorporated intoan appropriate pharmaceutical composition described herein andadministered to the subject through a route which allows the molecule,polypeptide, modulator, or compound etc. to perform its intendedfunction. Examples of routes of administration are also described hereinunder subsection IV.

[0160] d. Pharmacogenomics

[0161] Test/candidate compounds, or modulators which have a stimulatoryor inhibitory effect on mACHR-6 activity (e.g., mACHR-6 gene expression)as identified by a screening assay described herein can be administeredto individuals to treat (prophylactically or therapeutically) disorders(e.g., CNS disorders) associated with aberrant mACHR-6 activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permit the selection of effectivecompounds (e.g., drugs) for prophylactic or therapeutic treatments basedon a consideration of the individual's genotype. Such pharmacogenomicscan further be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of mACHR-6 polypeptide, expressionof mACHR-6 nucleic acid, or mutation content of mACHR-6 genes in anindividual can be determined to thereby select appropriate compound(s)for therapeutic or prophylactic treatment of the individual.

[0162] Pharmacogenomics deal with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, e.g., Eichelbaum, M. (1996)Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M. W.(1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raredefects or as polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

[0163] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0164] Thus, the activity of mACHR-6 polypeptide, expression of mACHR-6nucleic acid, or mutation content of mACHR-6 genes in an individual canbe determined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of a subject. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of a subject's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith an mACHR-6 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

[0165] e. Monitoring of Effects During Clinical Trials

[0166] Monitoring the influence of compounds (e.g., drugs) on theexpression or activity of mACHR-6 (e.g., the ability to modulate theeffects of acetylcholine on acetylcholine responsive cells) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay, as described herein, to increase mACHR-6 gene expression,polypeptide levels, or up-regulate mACHR-6 activity, can be monitored inclinical trails of subjects exhibiting decreased mACHR-6 geneexpression, polypeptide levels, or down-regulated mACHR-6 activity.Alternatively, the effectiveness of an agent, determined by a screeningassay, to decrease mACHR-6 gene expression, polypeptide levels, ordown-regulate mACHR-6 activity, can be monitored in clinical trails ofsubjects exhibiting increased mACHR-6 gene expression, polypeptidelevels, or up-regulated mACHR-6 activity. In such clinical trials, theexpression or activity of mACHR-6 and, preferably, other genes whichhave been implicated in, for example, a nervous system related disordercan be used as a “read out” or markers of the acetylcholineresponsiveness of a particular cell.

[0167] For example, and not by way of limitation, genes, includingmACHR-6, which are modulated in cells by treatment with a compound(e.g., drug or small molecule) which modulates mACHR-6 activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of compounds on CNS disorders, for example, ina clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of mACHR-6 and other genes implicated inthe disorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount ofpolypeptide produced, by one of the methods described herein, or bymeasuring the levels of activity of mACHR-6 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the compound. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the compound.

[0168] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject with acompound (e.g., an agonist, antagonist, peptidomimetic, polypeptide,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) comprising thesteps of (i) obtaining a pre-administration sample from a subject priorto administration of the compound; (ii) detecting the level ofexpression of an mACHR-6 polypeptide, mRNA, or genomic DNA in thepreadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the mACHR-6 polypeptide, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the mACHR-6 polypeptide, mRNA, or genomic DNAin the pre-administration sample with the mACHR-6 polypeptide, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the compound to the subject accordingly.For example, increased administration of the compound may be desirableto increase the expression or activity of mACHR-6 to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of mACHR-6 to lower levels thandetected, i.e. to decrease the effectiveness of the compound.

[0169] VI. Uses of Partial mACHR-6 Sequences

[0170] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (a) map their respective genes on a chromosome; and, thus,locate gene regions associated with genetic disease; (b) identify anindividual from a minute biological sample (tissue typing); and (c) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

[0171] a. Chromosome Mapping

[0172] Once the sequence (or a portion of the sequence) of a gene hasbeen isolated, this sequence can be used to map the location of the geneon a chromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the mACHR-6, sequences, described herein, canbe used to map the location of the mACHR-6 gene, respectively, on achromosome. The mapping of the mACHR-6 sequence to chromosomes is animportant first step in correlating these sequence with genes associatedwith disease.

[0173] Briefly, the mACHR-6 gene can be mapped to a chromosome bypreparing PCR primers (preferably)15-25 bp in length) from the mACHR-6sequence. Computer analysis of the mACHR-6, sequence can be used torapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primerscan then be used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the mACHR-6 sequence will yield an amplifiedfragment.

[0174] Somatic cell hybrids are prepared by fusing somatic cells fromdifferent mammals (e.g., human and mouse cells). As hybrids of human andmouse cells grow and divide, they gradually lose human chromosomes inrandom order, but retain the mouse chromosomes. By using media in whichmouse cells cannot grow, because they lack a particular enzyme, buthuman cells can, the one human chromosome that contains the geneencoding the needed enzyme, will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. (D'EustachioP. et al. (1983) Science 220:919-924). Somatic cell hybrids containingonly fragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

[0175] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular sequence to a particular chromosome. Three ormore sequences can be assigned per day using a single thermal cycler.Using the mACHR-6 sequence to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa mACHR-6 sequence to its chromosome include in situ hybridization(described in Fan, Y. et al. (1990) PNAS, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries.

[0176] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence-as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988).

[0177] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0178] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data (such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland, J. etal. (1987) Nature, 325:783-787.

[0179] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the mACHR-6 gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0180] b. Tissue Typing

[0181] The mACHR-6 sequences of the present invention can also be usedto identify individuals from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique, an individual's genomic DNA is digested with one ormore restriction enzymes, and probed on a Southern blot to yield uniquebands for identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

[0182] Furthermore, the sequences of the present invention can be usedto provide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the mACHR-6 sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

[0183] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The mACHR-6 sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NOs:1, 4, and 31, cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NOs:3, 6, and 33, are used, a more appropriate number of primersfor positive individual identification would be 500-2,000.

[0184] If a panel of reagents from mACHR-6 sequences described herein isused to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0185] c. Use of Partial mACHR-6 Sequences in Forensic Biology

[0186] DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

[0187] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As described above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NOs:1, 4, and 31 areparticularly appropriate for this use as greater numbers ofpolymorphisms occur in the noncoding regions, making it easier todifferentiate individuals using this technique. Examples ofpolynucleotide reagents include the mACHR-6 sequences or portionsthereof, e.g., fragments derived from the noncoding regions of SEQ IDNOs:1, 4, and 31, having a length of at least 20 bases, preferably atleast 30 bases.

[0188] The mACHR-6 sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such mACHR-6 probes can be used to identifytissue by species and/or by organ type.

[0189] In a similar fashion, these reagents, e.g., mACHR-6 primers orprobes can be used to screen tissue culture for contamination (i.e.screen for the presence of a mixture of different types of cells in aculture).

[0190] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patent applications, patents, and published patentapplications cited throughout this application are hereby incorporatedby reference.

EXAMPLES Example 1 Identification of Rat And Human mACHR-6 cDNA

[0191] In this example, mACHR-6 nucleic acid molecules were identifiedby screening appropriate cDNA libraries. More specifically, a ratfrontal cortex oligo dT-primed cDNA library was plated out and coloniespicked into 96 well plates. The colonies were cultured, plasmids wereprepared from each well, and the 5′ end of each insert sequenced. Afterautomated “trimming” of non-insert sequences, the nucleotide sequenceswere compared against the public protein databases using the BLASTsequence comparison program (BLASTN1.3MP, Altschul et al. (1990) J. Mol.Biol. 215:403). Upon review of the results from this sequencecomparison, a single clone was identified, designated 84g5, whosehighest similarity was with the rat muscarinic acetylcholine receptor M1(mACHR M1; GenBank™ Accession Number P08482). The clone containing thissequence was recovered from the 96 well plate, plasmid was preparedusing standard methods and the insert fully sequenced using standard“contigging” techniques. A repeat BLAST analysis using the entire insertsequence once again showed that the sequence in the protein databasewith the greatest similarity corresponded to GenBank™ Accession NumberP08482. This sequence and the insert sequence were compared using theGAP program in the GCG software package using a gap weight of 5.000 anda length weight of 0.100. The results showed a 27.97% identity and49.01% similarity between the two sequences with the insertion of 4 gapsfor optimized sequence alignment. The alignment indicated that the 84g5clone does not extend fully across the P08482 sequence, apparentlymissing approximately 30 amino acid residues at the N-terminal region ofthe molecule. A probe spanning residues 143-249 of SEQ ID NO:31 was thenused to re-screen the same frontal cortex library. This resulted in theindentification of the full length rat mACHR-6 sequence shown in SEQ IDNO:4 BLAST analysis of public nucleotide databases revealed noequivalent human sequences. Only a single mouse EST was identified(GenBank™ Accession Number AA118949) which is similar to the 84g5 clonebetween residues 1101 and 1650.

[0192] The human mACHR-6 nucleic acid molecule was identified byscreening a human cerebellum cDNA library using a Nci I/Not Irestriction fragment of the rat cDNA as a probe. BLAST analysis ofprotein and nucleic acid databases in the public domain again showedthat the mACHR-6 nucleic acid molecule is most similar to mACHR M1sequences. The alignments also revealed that mAChR-6 nucleic acidmolecule encodes a full length mACHR polypeptide.

Example 2 Tissue Expression of the mACHR-6 Gene

[0193] Northern Analysis Using RNA from Human and Rat Tissue

[0194] Human brain multiple tissue northern (MTN) blots, human MTN I,II, and III blots, and rat MTN blots (Clontech, Palo Alto, Calif.),containing 2 μg of poly A+ RNA per lane were probed with the rat mACHR-6nucleotide sequence (Nci I/Not I restriction fragment). The filters wereprehybridized in 10 ml of Express Hyb hybridization solution (Clontech,Palo Alto, Calif.) at 68° C. for 1 hour, after which 100 ng of ³²Plabeled probe was added. The probe was generated using the StratagenePrime-It kit, Catalog Number 300392 (Clontech, Palo Alto, Calif.).Hybridization was allowed to proceed at 68° C. for approximately 2hours. The filters were washed in a 0.05% SDS/2×SSC solution for 15minutes at room temperature and then twice with a 0.1% SDS/0.1×SSCsolution for 20 minutes at 50° C. and then exposed to autoradiographyfilm overnight at −80° C. with one screen. The human tissues testedincluded: heart, brain (regions of the brain tested included cerebellum,corpus callosum, cerebral cortex, medulla, occipital pole, frontal lobe,temporal lobe, putamen, amygdala, caudate nucleus, hippocampus,substantia nigra, subthalamic nucleus and thalamus), placenta, lung,liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,testis, ovary, small intestine, colon, peripheral blood leukocyte,stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland andbone marrow. The rat tissues tested included: heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis.

[0195] There was a strong hybridization to human whole brain, thefollowing human brain regions: cerebellum, corpus callosum, cerebralcortex, medulla, occipital pole, frontal lobe, temporal lobe, putamen,amygdala, caudate nucleus, hippocampus, substantia nigra, subthalamicnucleus and thalamus; and rat brain indicating that the approximately 3kb mACHR-6 gene transcript is expressed in these tissues. There was alsohybridization to human spinal cord.

[0196] In Situ Hybridization

[0197] For in situ analysis, the brain of an adult Sprague-Dawley ratwas removed and frozen on dry ice. Ten-micrometer-thick coronal sectionsof the brain were postfixed with 4% formaldehyde in DEPC treated1×phosphate-buffered saline at room temperature for 10 minutes beforebeing rinsed twice in DEPC 1×phosphate-buffered saline and once in 0.1 Mtriethanolamine-HCl (pH 8.0). Following incubation in 0.25% aceticanhydride-0.1 M triethanolamine-HCl for 10 minutes, sections were rinsedin DEPC 2×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissuewas then dehydrated through a series of ethanol washes, incubated in100% chloroform for 5 minutes, and then rinsed in 100% ethanol for 1minute and 95% ethanol for 1 minute and allowed to air dry.

[0198] Hybridizations were performed with ³⁵S-radiolabeled (5×10⁷cpm/ml) cRNA probes encoding a 474-bp fragment of the rat gene(generated with PCR primers F, 5′-CAAGAACCCMTTAAGCCAAG (SEQ ID NO:27),and R, 5′-GAAGAAGGTAACGCTGAGGA (SEQ ID NO:28)) and a 529-bp fragment ofthe rat gene (generated with PCR primers F, 5′-CAGAACCCCCACCAGATGCC (SEQID NO:29), and R, 5′-TAGTGGCACAGTGGGTAGAG (SEQ ID NO:30)). Probes wereincubated in the presence of a solution containing 600 mM NaCl, 10 mMTris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeasttRNA, 0.05% yeast total RNA type X1, 1× Denhardt's solution, 50%formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodiumdodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55°C.

[0199] After hybridization, slides were washed with 2×SSC. Sections werethen sequentially incubated at 37° C. in TNE (a solution containing 10mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, inTNE with 10 μg of RNase A per ml for 30 minutes, and finally in TNE for10 minutes. Slides were then rinsed with 2×SSC at room temperature,washed with 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C.for 1 hour, and 0.2×SSC at 60° C. for 1 hour. Sections were thendehydrated rapidly through serial ethanol-0.3 M sodium acetateconcentrations before being air dried and exposed to Kodak Biomax MRscientific imaging film for 24 hours and subsequently dipped in NB-2photoemulsion and exposed at 4° C. for 7 days before being developed andcounter stained.

[0200] Significant hybridization was seen in a number of brain regions.These included the cortex, caudate putamen, hippocampus, thalamus andcerebellum. Analysis of these regions at high magnification showed thatsignificant labeling was seen over the cell bodies of neurons.

Example 3 Expression of Recombinant mACHR-6 Polypeptide in BacterialCells

[0201] In this example, mACHR-6 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, mACHR-6is fused to GST and this fusion polypeptide is expressed in E. coli,e.g., strain PEB199. As the human and rat mACHR-6 polypeptides arepredicted to be approximately 51.3 kDa, and 51.2 kDa, respectively, andGST is predicted to be 26 kDa, the fusion polypeptides are predicted tobe approximately 77.3 kDa and 77.2 kDa, respectively, in molecularweight. Expression of the GST-mACHR-6 fusion polypeptide in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant mACHR-6 Polypeptide in COS Cells

[0202] To express the mACHR-6 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire mACHR-6 polypeptide and a HA tag (Wilson et al. (1984) Cell37:767) fused in-frame to its 3′ end of the fragment is cloned into thepolylinker region of the vector, thereby placing the expression of therecombinant polypeptide under the control of the CMV promoter.

[0203] To construct the plasmid, the mACHR-6 DNA sequence is amplifiedby PCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the mACHR-6coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag and the last 20nucleotides of the mACHR-6 coding sequence. The PCR amplified fragmentand the pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the mACHR-6 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

[0204] COS cells are subsequently transfected with the mACHR-6-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the mACHR-6 polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory-Press, Cold Spring Harbor, N.Y., 1988) using an HA specificmonoclonal antibody. Briefly, the cells are labelled for 8 hours with³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collectedand the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate andthe culture media are precipitated with an HA specific monoclonalantibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[0205] Alternatively, DNA containing the mACHR-6 coding sequence iscloned directly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of themACHR-6 polypeptide is detected by radiolabelling andimmunoprecipitation using an mACHR-6 specific monoclonal antibody.

Example 5 Characterization of the Human and Rat mACHR-6 Polypeptides

[0206] In this example, the amino acid sequences of the human and therat mACHR-6 polypeptides were compared to amino acid sequences of knownpolypeptides and various motifs were identified.

[0207] The human mACHR-6 polypeptide, the amino acid sequence of whichis shown in FIG. 1 (SEQ ID NO:2), is a novel polypeptide which includes445 amino acid residues. The human mACHR-6 polypeptide contains seventransmembrane domains between amino acid residues 34-59 (SEQ ID NO:7),73-91 (SEQ ID NO:8), 109-130 (SEQ ID NO:9), 152-174 (SEQ ID NO:10),197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and 396-416 (SEQ IDNO:13). The nucleotide sequence of the human mACHR-6 was used as adatabase query using the BLASTN program (BLASTN1.3 MP, Altschul et al.(1990) J. Mol. Biol. 215:403). The closest hits were human, rat, mouseand pig mACHR M1 (GenBank™ Accession Numbers P11229, P08482, P12657, andP04761, respectively). The highest similarity is 32/70 amino acididentities.

[0208] The rat mACHR-6 polypeptide, the amino acid sequence of which isshown in FIG. 2 (SEQ ID NO:5), is a novel polypeptide which includes 445amino acid residues. The rat mACHR-6 polypeptide contains seventransmembrane domains between amino acid residues 34-59 (SEQ ID NO:14),73-91 (SEQ ID NO:15), 109-130 (SEQ ID NO:16), 152-174 (SEQ ID NO:17),197-219 (SEQ ID NO:18), 360-380 (SEQ ID NO:19) and 396416 (SEQ IDNO:20), which correspond to the human mACHR-6 polypeptide transmembranedomains 1-7 (SEQ ID NOs:7-13). The nucleotide sequence of the ratmACHR-6 was used as a database query using the BLASTN program (BLASTN1.3MP, Altschul et al. (1990) J. Mol. Biol. 215:403) The closest hits werehuman, rat, mouse and pig mACHR M1 (GenBank™ Accession Numbers P11229,P08482, P12657, and P04761, respectively). The highest similarity is33/70 amino acid identities. Hydropathy plots indicated that thetransmembrane domains of the rat mACHR-6 polypeptide are similar tothose of the rat mACHR M1. The cysteines (residues 63 and 44 of SEQ IDNO:5) that give rise to intramolecular disulfide bonds are alsoconserved.

[0209] Equivalents

[0210] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 39 2689 base pairs nucleic acid single linear cDNA CDS 291..1625 1GTCGACCCAC GCGTCCGCGC ACCGGCAGCG GCTCAGGCTC CGGCTCCTCT CCCGCTGCAG 60CAGCCGCGCT GCCGGCCCCA CTGGGCTCGG ATCCGGCCCC GGCCCCCTCG GCACCGCCTG 120CTCTGGCCCC GGCCCCGGCC CCGCGGACCA TGCGCTGGGC GCCCCCAGGG GAACCCGACC 180CGGCCAAGGG CCCGCAAAGA CGAGGCTCCC GGGCCGGGGC CCCTCCCGGC CGCCCAGCTC 240TCGGCCGGCG CCCTGCCCCG CGTCCCGGAG CCGCGTGAGC CTGCGGGGCC ATG GAG 296 MetGlu 1 CGC GCG CCG CCC GAC GGG CCG CTG AAC GCT TCG GGG GCG CTG GCG GGC344 Arg Ala Pro Pro Asp Gly Pro Leu Asn Ala Ser Gly Ala Leu Ala Gly 5 1015 GAG GCG GCG GCG GCG GGC GGG GCG CGC GGC TTC TCG GCA GCC TGG ACC 392Glu Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala Trp Thr 20 25 30GCG GTG CTG GCC GCG CTC ATG GCG CTG CTC ATC GTG GCC ACG GTG CTG 440 AlaVal Leu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr Val Leu 35 40 45 50GGC AAC GCG CTG GTC ATG CTC GCC TTC GTG GCC GAC TCG AGC CTC CGC 488 GlyAsn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser Leu Arg 55 60 65 ACCCAG AAC AAC TTC TTC CTG CTC AAC CTC GCC ATC TCC GAC TTC CTC 536 Thr GlnAsn Asn Phe Phe Leu Leu Asn Leu Ala Ile Ser Asp Phe Leu 70 75 80 GTC GGCGCC TTC TGC ATC CCA CTG TAT GTA CCC TAC GTG CTG ACA GGC 584 Val Gly AlaPhe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu Thr Gly 85 90 95 CGC TGG ACCTTC GGC CGG GGC CTC TGC AAG CTG TGG CTG GTA GTG GAC 632 Arg Trp Thr PheGly Arg Gly Leu Cys Lys Leu Trp Leu Val Val Asp 100 105 110 TAC CTG CTGTGC ACC TCC TCT GCC TTC AAC ATC GTG CTC ATC AGC TAC 680 Tyr Leu Leu CysThr Ser Ser Ala Phe Asn Ile Val Leu Ile Ser Tyr 115 120 125 130 GAC CGCTTC CTG TCG GTC ACC CGA GCG GTC TCA TAC CGG GCC CAG CAG 728 Asp Arg PheLeu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala Gln Gln 135 140 145 GGT GACACG CGG CGG GCA GTG CGG AAG ATG CTG CTG GTG TGG GTG CTG 776 Gly Asp ThrArg Arg Ala Val Arg Lys Met Leu Leu Val Trp Val Leu 150 155 160 GCC TTCCTG CTG TAC GGA CCA GCC ATC CTG AGC TGG GAG TAC CTG TCC 824 Ala Phe LeuLeu Tyr Gly Pro Ala Ile Leu Ser Trp Glu Tyr Leu Ser 165 170 175 GGG GGCAGC TCC ATC CCC GAG GGC CAC TGC TAT GCC GAG TTC TTC TAC 872 Gly Gly SerSer Ile Pro Glu Gly His Cys Tyr Ala Glu Phe Phe Tyr 180 185 190 AAC TGGTAC TTC CTC ATC ACG GCT TCC ACC CTG GAG TTC TTT ACG CCC 920 Asn Trp TyrPhe Leu Ile Thr Ala Ser Thr Leu Glu Phe Phe Thr Pro 195 200 205 210 TTCCTC AGC GTC ACC TTC TTT AAC CTC AGC ATC TAC CTG AAC ATC CAG 968 Phe LeuSer Val Thr Phe Phe Asn Leu Ser Ile Tyr Leu Asn Ile Gln 215 220 225 AGGCGC ACC CGC CTC CGG CTG GAT GGG GCT CGA GAG GCA GCC GGC CCC 1016 Arg ArgThr Arg Leu Arg Leu Asp Gly Ala Arg Glu Ala Ala Gly Pro 230 235 240 GAGCCC CCT CCC GAG GCC CAG CCC TCA CCA CCC CCA CCG CCT GGC TGC 1064 Glu ProPro Pro Glu Ala Gln Pro Ser Pro Pro Pro Pro Pro Gly Cys 245 250 255 TGGGGC TGC TGG CAG AAG GGG CAC GGG GAG GCC ATG CCG CTG CAC AGG 1112 Trp GlyCys Trp Gln Lys Gly His Gly Glu Ala Met Pro Leu His Arg 260 265 270 TATGGG GTG GGT GAG GCG GCC GTA GGC GCT GAG GCC GGG GAG GCG ACC 1160 Tyr GlyVal Gly Glu Ala Ala Val Gly Ala Glu Ala Gly Glu Ala Thr 275 280 285 290CTC GGG GGT GGC GGT GGG GGC GGC TCC GTG GCT TCA CCC ACC TCC AGC 1208 LeuGly Gly Gly Gly Gly Gly Gly Ser Val Ala Ser Pro Thr Ser Ser 295 300 305TCC GGC AGC TCC TCG AGG GGC ACT GAG AGG CCG CGC TCA CTC AAG AGG 1256 SerGly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu Lys Arg 310 315 320GGC TCC AAG CCG TCG GCG TCC TCG GCC TCA CTG GAG AAG CGC ATG AAG 1304 GlySer Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg Met Lys 325 330 335ATG GTG TCC CAG AGC TTC ACC CAG CGC TTT CGG CTG TCT CGG GAC AGG 1352 MetVal Ser Gln Ser Phe Thr Gln Arg Phe Arg Leu Ser Arg Asp Arg 340 345 350AAA GTG GCC AAG TCG CTG GCC GTC ATC GTG AGC ATC TTT GGG CTC TGC 1400 LysVal Ala Lys Ser Leu Ala Val Ile Val Ser Ile Phe Gly Leu Cys 355 360 365370 TGG GCC CCA TAC ACG CTG CTG ATG ATC ATC CGG GCC GCC TGC CAT GGC 1448Trp Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys His Gly 375 380385 CAC TGC GTC CCT GAC TAC TGG TAC GAA ACC TCC TTC TGG CTC CTG TGG 1496His Cys Val Pro Asp Tyr Trp Tyr Glu Thr Ser Phe Trp Leu Leu Trp 390 395400 GCC AAC TCG GCT GTC AAC CCT GTC CTC TAC CCT CTG TGC CAC CAC AGC 1544Ala Asn Ser Ala Val Asn Pro Val Leu Tyr Pro Leu Cys His His Ser 405 410415 TTC CGC CGG GCC TTC ACC AAG CTG CTC TGC CCC CAG AAG CTC AAA ATC 1592Phe Arg Arg Ala Phe Thr Lys Leu Leu Cys Pro Gln Lys Leu Lys Ile 420 425430 CAG CCC CAC AGC TCC CTG GAG CAC TGC TGG AAG TGAGTGGCCC ACCAGAGCCT1645 Gln Pro His Ser Ser Leu Glu His Cys Trp Lys 435 440 445 CCCTCAGCCACGCCTCTCTC AGCCCAGGTC TCCTGGGCAT CTGGCCCTGC TGCCCCCTAC 1705 CCGGCTCGTTCCCCCAGGGG TGAGCCCCGC CGTGTCTGTG GCCCTCTCTT AATGCCACGG 1765 CAGCCACCCTGCCATGGAGG CGCCTTCCTG GGTTGGCCAG AGGGCCCCTC ACTGGCTGGA 1825 CTGGAGGCTGGGTGGCCGGC CCTGCCCCCC ACATTCTGGC TCCACCGGGA GGGACAGTCT 1885 GGAGGTCCCAGACATGCTGC CCACCCCCTG CTGGTGCCCA CCCTTCGCAG TTACTGGTTG 1945 GTGTTCTTCCCAAAGCAAGC ACCTGGGTGT GCTCCAGGCT TCCTGCCCTA GCAGTTTGCC 2005 TCTGCACGTGCACACACCTG CACACCCCTG CACACACCTG CACACCGTCC CTCTCCCCGG 2065 ACAAGCCCAGGACACTGCCT TTGCTGCCTT CTGTCTCTTG CATAAGCCTC AGGCCTGGCC 2125 CTTTCACCCCTCTTCCCACC AACTCTCTCT GCCCCCAAAA GTGTCAAGGG GCCCTAGGAA 2185 CCTCGAAGCTGTTCTCTGCT TTTCCATTCT GGGTGTTTTC AGAAAGATGA AGAAGAAAAC 2245 ATGTCTGTGAACTTGATGTT CCTGGGATGT TTAATCAAGA GAGACAAAAT TGCTGAGGAG 2305 CTCAGGGCTGGATTGGCAGG TGTGGGCTCC CACGCCCTCC TCCCTCCGCT AAGGCTTCCG 2365 GCTGAGCTGTGCCAGCTGCT TCTGCCCACC CCGCCTCTGG GCTCACACCA GCCCTGGTGG 2425 CCAAGCCTGCCCCGGCCACT CTGTTTGCTC ACCCAGGACC TCTGGGGGTT GTTGGGAGGA 2485 GGGGGCCCGGCTGGGCCCGA GGGTCCCAAG GCGTGCAGGG GCGGTCCAGA GGAGGTGCCC 2545 GGGCAGGGGCCGCTTCGCCA TGTGCTGTGC ACCCGTGCCA CGCGCTCTGC ATGCTCCTCT 2605 GCCTGTGCCCGCTGCGCTGC CCTGCAAACC GTGAGGTCAC AATAAAGTGT ATTTTTTTAA 2665 AAAAAAAAAAAAAAGGGCGG CCGC 2689 445 amino acids amino acid linear protein 2 Met GluArg Ala Pro Pro Asp Gly Pro Leu Asn Ala Ser Gly Ala Leu 1 5 10 15 AlaGly Glu Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala 20 25 30 TrpThr Ala Val Leu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr 35 40 45 ValLeu Gly Asn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser 50 55 60 LeuArg Thr Gln Asn Asn Phe Phe Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80Phe Leu Val Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu 85 90 95Thr Gly Arg Trp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val 100 105110 Val Asp Tyr Leu Leu Cys Thr Ser Ser Ala Phe Asn Ile Val Leu Ile 115120 125 Ser Tyr Asp Arg Phe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala130 135 140 Gln Gln Gly Asp Thr Arg Arg Ala Val Arg Lys Met Leu Leu ValTrp 145 150 155 160 Val Leu Ala Phe Leu Leu Tyr Gly Pro Ala Ile Leu SerTrp Glu Tyr 165 170 175 Leu Ser Gly Gly Ser Ser Ile Pro Glu Gly His CysTyr Ala Glu Phe 180 185 190 Phe Tyr Asn Trp Tyr Phe Leu Ile Thr Ala SerThr Leu Glu Phe Phe 195 200 205 Thr Pro Phe Leu Ser Val Thr Phe Phe AsnLeu Ser Ile Tyr Leu Asn 210 215 220 Ile Gln Arg Arg Thr Arg Leu Arg LeuAsp Gly Ala Arg Glu Ala Ala 225 230 235 240 Gly Pro Glu Pro Pro Pro GluAla Gln Pro Ser Pro Pro Pro Pro Pro 245 250 255 Gly Cys Trp Gly Cys TrpGln Lys Gly His Gly Glu Ala Met Pro Leu 260 265 270 His Arg Tyr Gly ValGly Glu Ala Ala Val Gly Ala Glu Ala Gly Glu 275 280 285 Ala Thr Leu GlyGly Gly Gly Gly Gly Gly Ser Val Ala Ser Pro Thr 290 295 300 Ser Ser SerGly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu 305 310 315 320 LysArg Gly Ser Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg 325 330 335Met Lys Met Val Ser Gln Ser Phe Thr Gln Arg Phe Arg Leu Ser Arg 340 345350 Asp Arg Lys Val Ala Lys Ser Leu Ala Val Ile Val Ser Ile Phe Gly 355360 365 Leu Cys Trp Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys370 375 380 His Gly His Cys Val Pro Asp Tyr Trp Tyr Glu Thr Ser Phe TrpLeu 385 390 395 400 Leu Trp Ala Asn Ser Ala Val Asn Pro Val Leu Tyr ProLeu Cys His 405 410 415 His Ser Phe Arg Arg Ala Phe Thr Lys Leu Leu CysPro Gln Lys Leu 420 425 430 Lys Ile Gln Pro His Ser Ser Leu Glu His CysTrp Lys 435 440 445 1335 base pairs nucleic acid single linear cDNA CDS1..1335 3 ATG GAG CGC GCG CCG CCC GAC GGG CCG CTG AAC GCT TCG GGG GCGCTG 48 Met Glu Arg Ala Pro Pro Asp Gly Pro Leu Asn Ala Ser Gly Ala Leu 15 10 15 GCG GGC GAG GCG GCG GCG GCG GGC GGG GCG CGC GGC TTC TCG GCA GCC96 Ala Gly Glu Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala 20 2530 TGG ACC GCG GTG CTG GCC GCG CTC ATG GCG CTG CTC ATC GTG GCC ACG 144Trp Thr Ala Val Leu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr 35 40 45GTG CTG GGC AAC GCG CTG GTC ATG CTC GCC TTC GTG GCC GAC TCG AGC 192 ValLeu Gly Asn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser 50 55 60 CTCCGC ACC CAG AAC AAC TTC TTC CTG CTC AAC CTC GCC ATC TCC GAC 240 Leu ArgThr Gln Asn Asn Phe Phe Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 TTCCTC GTC GGC GCC TTC TGC ATC CCA CTG TAT GTA CCC TAC GTG CTG 288 Phe LeuVal Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu 85 90 95 ACA GGCCGC TGG ACC TTC GGC CGG GGC CTC TGC AAG CTG TGG CTG GTA 336 Thr Gly ArgTrp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val 100 105 110 GTG GACTAC CTG CTG TGC ACC TCC TCT GCC TTC AAC ATC GTG CTC ATC 384 Val Asp TyrLeu Leu Cys Thr Ser Ser Ala Phe Asn Ile Val Leu Ile 115 120 125 AGC TACGAC CGC TTC CTG TCG GTC ACC CGA GCG GTC TCA TAC CGG GCC 432 Ser Tyr AspArg Phe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala 130 135 140 CAG CAGGGT GAC ACG CGG CGG GCA GTG CGG AAG ATG CTG CTG GTG TGG 480 Gln Gln GlyAsp Thr Arg Arg Ala Val Arg Lys Met Leu Leu Val Trp 145 150 155 160 GTGCTG GCC TTC CTG CTG TAC GGA CCA GCC ATC CTG AGC TGG GAG TAC 528 Val LeuAla Phe Leu Leu Tyr Gly Pro Ala Ile Leu Ser Trp Glu Tyr 165 170 175 CTGTCC GGG GGC AGC TCC ATC CCC GAG GGC CAC TGC TAT GCC GAG TTC 576 Leu SerGly Gly Ser Ser Ile Pro Glu Gly His Cys Tyr Ala Glu Phe 180 185 190 TTCTAC AAC TGG TAC TTC CTC ATC ACG GCT TCC ACC CTG GAG TTC TTT 624 Phe TyrAsn Trp Tyr Phe Leu Ile Thr Ala Ser Thr Leu Glu Phe Phe 195 200 205 ACGCCC TTC CTC AGC GTC ACC TTC TTT AAC CTC AGC ATC TAC CTG AAC 672 Thr ProPhe Leu Ser Val Thr Phe Phe Asn Leu Ser Ile Tyr Leu Asn 210 215 220 ATCCAG AGG CGC ACC CGC CTC CGG CTG GAT GGG GCT CGA GAG GCA GCC 720 Ile GlnArg Arg Thr Arg Leu Arg Leu Asp Gly Ala Arg Glu Ala Ala 225 230 235 240GGC CCC GAG CCC CCT CCC GAG GCC CAG CCC TCA CCA CCC CCA CCG CCT 768 GlyPro Glu Pro Pro Pro Glu Ala Gln Pro Ser Pro Pro Pro Pro Pro 245 250 255GGC TGC TGG GGC TGC TGG CAG AAG GGG CAC GGG GAG GCC ATG CCG CTG 816 GlyCys Trp Gly Cys Trp Gln Lys Gly His Gly Glu Ala Met Pro Leu 260 265 270CAC AGG TAT GGG GTG GGT GAG GCG GCC GTA GGC GCT GAG GCC GGG GAG 864 HisArg Tyr Gly Val Gly Glu Ala Ala Val Gly Ala Glu Ala Gly Glu 275 280 285GCG ACC CTC GGG GGT GGC GGT GGG GGC GGC TCC GTG GCT TCA CCC ACC 912 AlaThr Leu Gly Gly Gly Gly Gly Gly Gly Ser Val Ala Ser Pro Thr 290 295 300TCC AGC TCC GGC AGC TCC TCG AGG GGC ACT GAG AGG CCG CGC TCA CTC 960 SerSer Ser Gly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu 305 310 315320 AAG AGG GGC TCC AAG CCG TCG GCG TCC TCG GCC TCA CTG GAG AAG CGC 1008Lys Arg Gly Ser Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg 325 330335 ATG AAG ATG GTG TCC CAG AGC TTC ACC CAG CGC TTT CGG CTG TCT CGG 1056Met Lys Met Val Ser Gln Ser Phe Thr Gln Arg Phe Arg Leu Ser Arg 340 345350 GAC AGG AAA GTG GCC AAG TCG CTG GCC GTC ATC GTG AGC ATC TTT GGG 1104Asp Arg Lys Val Ala Lys Ser Leu Ala Val Ile Val Ser Ile Phe Gly 355 360365 CTC TGC TGG GCC CCA TAC ACG CTG CTG ATG ATC ATC CGG GCC GCC TGC 1152Leu Cys Trp Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys 370 375380 CAT GGC CAC TGC GTC CCT GAC TAC TGG TAC GAA ACC TCC TTC TGG CTC 1200His Gly His Cys Val Pro Asp Tyr Trp Tyr Glu Thr Ser Phe Trp Leu 385 390395 400 CTG TGG GCC AAC TCG GCT GTC AAC CCT GTC CTC TAC CCT CTG TGC CAC1248 Leu Trp Ala Asn Ser Ala Val Asn Pro Val Leu Tyr Pro Leu Cys His 405410 415 CAC AGC TTC CGC CGG GCC TTC ACC AAG CTG CTC TGC CCC CAG AAG CTC1296 His Ser Phe Arg Arg Ala Phe Thr Lys Leu Leu Cys Pro Gln Lys Leu 420425 430 AAA ATC CAG CCC CAC AGC TCC CTG GAG CAC TGC TGG AAG 1335 Lys IleGln Pro His Ser Ser Leu Glu His Cys Trp Lys 435 440 445 3244 base pairsnucleic acid single linear cDNA CDS 778..2112 4 TGGCACGACA GGTTTCCCGACTGGAAAGCG GGCAGTGAGC GCAACGCAAT TAATGTGAGT 60 TAGCTCACTC ATTAGGCACCCCAGGCTTTA CACTTTATGC TTCCGGCTCG TATGTTGTGT 120 GGAATTGTGA GCGGATAACAATTTCACACA GGAAACAGCT ATGACCATGA TTACGCCAAG 180 CTTGGTACCG AGCTCGGATCCACTAGTAAC GGCCGCCAGT GTGCTGGAAT TCGGCTTGCG 240 GGCAGTGAGC GCAACGCAATTAATGTGAGT TAGCTCACTC ATTAGGCACC CCAGGCTTTA 300 CACTTTATGC TTCCGGCTCGTATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA 360 GGAAACAGCT ATGACCATGATTACGCCAAG CTCTAATACG ACTCACTATA GGGAAAGCTG 420 GTACGCCTGC AGGTACCGGTCCGGAATTCC CGGGTCGACC CACGCGCCCG CGCTGAGCTA 480 GGGGTGCACC GACGCACCGCGGGCGGCTGG AGCTCGGCTT TGCTCTCGCT GCAGCAGCCG 540 CGCCGCCCGC CCCACTCCGCTCAGATTCCG ACACCAGCCC CCTCTGGATC GCCCTCCTGG 600 ACTCTAGCCC GGGCTCTTGCTCCGACCCCG CGGACCATGC TCCGGGCGCC CCCCGGAAAA 660 CCGGGCTGGG CGAAGAGCCGGCAAAGATTA GGCTCACGAG CGGGGGCCCC ACCCGGCCAC 720 CCAGCTCTCC GCCCGTGCCCTGCCCGGTGT CCCCGAGCCG TGTGAGCCTG CTGGGCC 777 ATG GAG CGC GCG CCG CCC GACGGG CTG ATG AAC GCG TCG GGC ACT CTG 825 Met Glu Arg Ala Pro Pro Asp GlyLeu Met Asn Ala Ser Gly Thr Leu 1 5 10 15 GCC GGA GAG GCG GCG GCT GCAGGC GGG GCG CGC GGC TTC TCG GCT GCC 873 Ala Gly Glu Ala Ala Ala Ala GlyGly Ala Arg Gly Phe Ser Ala Ala 20 25 30 TGG ACC GCT GTC CTG GCT GCG CTCATG GCG CTG CTC ATC GTG GCC ACA 921 Trp Thr Ala Val Leu Ala Ala Leu MetAla Leu Leu Ile Val Ala Thr 35 40 45 GTA CTG GGC AAC GCG CTG GTC ATG CTCGCC TTC GTG GCG GAT TCG AGC 969 Val Leu Gly Asn Ala Leu Val Met Leu AlaPhe Val Ala Asp Ser Ser 50 55 60 CTC CGC ACC CAG AAC AAC TTC TTT CTG CTCAAC CTC GCC ATC TCC GAC 1017 Leu Arg Thr Gln Asn Asn Phe Phe Leu Leu AsnLeu Ala Ile Ser Asp 65 70 75 80 TTC CTC GTG GGT GCC TTC TGC ATC CCA TTGTAC GTA CCC TAT GTG CTG 1065 Phe Leu Val Gly Ala Phe Cys Ile Pro Leu TyrVal Pro Tyr Val Leu 85 90 95 ACC GGC CGT TGG ACC TTC GGC CGG GGC CTC TGCAAG CTG TGG CTG GTG 1113 Thr Gly Arg Trp Thr Phe Gly Arg Gly Leu Cys LysLeu Trp Leu Val 100 105 110 GTA GAC TAC CTA CTG TGT GCC TCC TCG GTC TTCAAC ATC GTA CTC ATC 1161 Val Asp Tyr Leu Leu Cys Ala Ser Ser Val Phe AsnIle Val Leu Ile 115 120 125 AGC TAT GAC CGA TTC CTG TCA GTC ACT CGA GCTGTC TCC TAC AGG GCC 1209 Ser Tyr Asp Arg Phe Leu Ser Val Thr Arg Ala ValSer Tyr Arg Ala 130 135 140 CAG CAG GGG GAC ACG AGA CGG GCC GTT CGG AAGATG GCA CTG GTG TGG 1257 Gln Gln Gly Asp Thr Arg Arg Ala Val Arg Lys MetAla Leu Val Trp 145 150 155 160 GTG CTG GCC TTC CTG CTG TAT GGG CCT GCCATC CTG AGT TGG GAG TAC 1305 Val Leu Ala Phe Leu Leu Tyr Gly Pro Ala IleLeu Ser Trp Glu Tyr 165 170 175 CTG TCT GGT GGC AGT TCC ATC CCC GAG GGCCAC TGC TAT GCT GAG TTC 1353 Leu Ser Gly Gly Ser Ser Ile Pro Glu Gly HisCys Tyr Ala Glu Phe 180 185 190 TTC TAC AAC TGG TAC TTT CTC ATC ACG GCCTCC ACC CTC GAG TTC TTC 1401 Phe Tyr Asn Trp Tyr Phe Leu Ile Thr Ala SerThr Leu Glu Phe Phe 195 200 205 ACG CCC TTC CTC AGC GTT ACC TTC TTC AACCTC AGC ATC TAC CTG AAC 1449 Thr Pro Phe Leu Ser Val Thr Phe Phe Asn LeuSer Ile Tyr Leu Asn 210 215 220 ATC CAG AGG CGC ACC CGC CTT CGG CTT GATGGG GGC CGT GAG GCT GGC 1497 Ile Gln Arg Arg Thr Arg Leu Arg Leu Asp GlyGly Arg Glu Ala Gly 225 230 235 240 CCA GAA CCC CCA CCA GAT GCC CAG CCCTCG CCA CCT CCA GCT CCC CCC 1545 Pro Glu Pro Pro Pro Asp Ala Gln Pro SerPro Pro Pro Ala Pro Pro 245 250 255 AGC TGC TGG GGC TGC TGG CCA AAA GGGCAT GGC GAG GCC ATG CCG TTG 1593 Ser Cys Trp Gly Cys Trp Pro Lys Gly HisGly Glu Ala Met Pro Leu 260 265 270 CAC AGG TAT GGG GTG GGT GAG GCA GGCCCT GGT GTT GAG GCT GGG GAG 1641 His Arg Tyr Gly Val Gly Glu Ala Gly ProGly Val Glu Ala Gly Glu 275 280 285 GCT GCC CTC GGG GGT GGC AGT GGT GGAGGT GCT GCT GCC TCG CCC ACC 1689 Ala Ala Leu Gly Gly Gly Ser Gly Gly GlyAla Ala Ala Ser Pro Thr 290 295 300 TCC AGC TCT GGC AGC TCC TCA AGG GGCACT GAG AGG CCA CGC TCA CTC 1737 Ser Ser Ser Gly Ser Ser Ser Arg Gly ThrGlu Arg Pro Arg Ser Leu 305 310 315 320 AAA AGG GGC TCC AAG CCA TCA GCATCT TCA GCA TCC CTG GAG AAG CGC 1785 Lys Arg Gly Ser Lys Pro Ser Ala SerSer Ala Ser Leu Glu Lys Arg 325 330 335 ATG AAG ATG GTG TCC CAG AGC ATCACC CAG CGC TTC CGG CTG TCG CGG 1833 Met Lys Met Val Ser Gln Ser Ile ThrGln Arg Phe Arg Leu Ser Arg 340 345 350 GAC AAG AAG GTG GCC AAG TCG CTGGCC ATC ATC GTG AGC ATC TTT GGG 1881 Asp Lys Lys Val Ala Lys Ser Leu AlaIle Ile Val Ser Ile Phe Gly 355 360 365 CTC TGC TGG GCG CCG TAC ACG CTCCTA ATG ATC ATC CGA GCT GCT TGC 1929 Leu Cys Trp Ala Pro Tyr Thr Leu LeuMet Ile Ile Arg Ala Ala Cys 370 375 380 CAT GGC CGC TGC ATC CCC GAT TACTGG TAC GAG ACG TCC TTC TGG CTT 1977 His Gly Arg Cys Ile Pro Asp Tyr TrpTyr Glu Thr Ser Phe Trp Leu 385 390 395 400 CTG TGG GCC AAC TCG GCC GTCAAC CCC GTC CTC TAC CCA CTG TGC CAC 2025 Leu Trp Ala Asn Ser Ala Val AsnPro Val Leu Tyr Pro Leu Cys His 405 410 415 TAC AGC TTC CGC AGA GCC TTCACC AAG CTC CTC TGC CCC CAG AAG CTC 2073 Tyr Ser Phe Arg Arg Ala Phe ThrLys Leu Leu Cys Pro Gln Lys Leu 420 425 430 AAG GTC CAG CCC CAC GGC TCCCTG GAG CAG TGC TGG AAG TGAGCAGCTG 2122 Lys Val Gln Pro His Gly Ser LeuGlu Gln Cys Trp Lys 435 440 445 CCCCACCCTT CTGAGGCCAG GCCCTTGTACTTGTTTGAGT GGGCAGCCGG AGCGTGGGCG 2182 GGGCCCTGGT CCATGCTCCG CTCCAAATGCCATGGCGGCC TCTTAGATCA TCAACCCCGC 2242 AGTGGGGTAG CATGGCAGGT GGGCCAAGAGCCCTAGTTGG TGGAGCTAGA GTGTGCTGGT 2302 TAGCTCTGCC GCCACATTCT CCTTCACCACACAGAAGAGA CAATCCAGGA GTCCCAGGCA 2362 TGCCTTCCAC CTACACACAC ACACACACACACACACACAC ACACACCACA GTGCAGTGCC 2422 AGTGATGTCC CCTTTTGCAT ATTTAGTGGTTGGTGTCCTC CCTAATGCAA ACCTCGGTGT 2482 GTGCTCCCGG CTCCGGCCCT GGCAATGCGTGCGTGCGCCC TGCATGTGCT CACACCCGCC 2542 ACACACCCGC CCGCCACACA CTTGCAACACCTCCTCTCTC CCAGAAGAGC TGGGGACGAT 2602 GCCCTTTGCT GCCACTGTCT CTTGCTTAATCCCAGAGCCT GGCTCCTTAT CCCCCACTCT 2662 CCCTTCAACT CTGCCCCACA AAGTGTCGAGCGCCTCGGGA AACTTGAAGC TTCTCTGCTC 2722 CTTCCACTCT GGATGTTTTC AGGAAGATGGAGGAGAAGAA AACACGTCTG TGAACTTGAT 2782 GTTCCTTGGA TGTTTAATCA AGAGAGACAAAATTGCCGAG GAGCTCGGGG CTGGATTGGC 2842 AGGTGTGGGC TCCCACGCCC TCCTCCCTCAGTGCTGCAGC TTCCGGCTGA GCCGCGCCAG 2902 CTGCTTCTGC CTGCCCCGCC CCCAGGCTTGGGACGATGGC CCTGCCCTGC TTGCCCCGTC 2962 TGTACAATCA GAATTTGGGG GTGGGTGGTTATGGGGTAGA GCGGCTCTTC ACTGTGCCCT 3022 AAAGGTCCTG AGGCTCACAG GACAGTCAGCAGGAGAGCAG GCAGGCCCGC GACACCTGGG 3082 AGGAATGCTT TGCCTCGTCC TGTGTACTCACCTCAGGCTT CTGCATGCTC TGCTGCCCTT 3142 GTGCCCTGGT GTGCTGCCTC TGCCAATGTGAAAACACAAT AAAGTGTATT TTTTTAAAAA 3202 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAGGGCGGCC GC 3244 445 amino acids amino acid linear protein 5 Met GluArg Ala Pro Pro Asp Gly Leu Met Asn Ala Ser Gly Thr Leu 1 5 10 15 AlaGly Glu Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala 20 25 30 TrpThr Ala Val Leu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr 35 40 45 ValLeu Gly Asn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser 50 55 60 LeuArg Thr Gln Asn Asn Phe Phe Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80Phe Leu Val Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu 85 90 95Thr Gly Arg Trp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val 100 105110 Val Asp Tyr Leu Leu Cys Ala Ser Ser Val Phe Asn Ile Val Leu Ile 115120 125 Ser Tyr Asp Arg Phe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala130 135 140 Gln Gln Gly Asp Thr Arg Arg Ala Val Arg Lys Met Ala Leu ValTrp 145 150 155 160 Val Leu Ala Phe Leu Leu Tyr Gly Pro Ala Ile Leu SerTrp Glu Tyr 165 170 175 Leu Ser Gly Gly Ser Ser Ile Pro Glu Gly His CysTyr Ala Glu Phe 180 185 190 Phe Tyr Asn Trp Tyr Phe Leu Ile Thr Ala SerThr Leu Glu Phe Phe 195 200 205 Thr Pro Phe Leu Ser Val Thr Phe Phe AsnLeu Ser Ile Tyr Leu Asn 210 215 220 Ile Gln Arg Arg Thr Arg Leu Arg LeuAsp Gly Gly Arg Glu Ala Gly 225 230 235 240 Pro Glu Pro Pro Pro Asp AlaGln Pro Ser Pro Pro Pro Ala Pro Pro 245 250 255 Ser Cys Trp Gly Cys TrpPro Lys Gly His Gly Glu Ala Met Pro Leu 260 265 270 His Arg Tyr Gly ValGly Glu Ala Gly Pro Gly Val Glu Ala Gly Glu 275 280 285 Ala Ala Leu GlyGly Gly Ser Gly Gly Gly Ala Ala Ala Ser Pro Thr 290 295 300 Ser Ser SerGly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu 305 310 315 320 LysArg Gly Ser Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg 325 330 335Met Lys Met Val Ser Gln Ser Ile Thr Gln Arg Phe Arg Leu Ser Arg 340 345350 Asp Lys Lys Val Ala Lys Ser Leu Ala Ile Ile Val Ser Ile Phe Gly 355360 365 Leu Cys Trp Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys370 375 380 His Gly Arg Cys Ile Pro Asp Tyr Trp Tyr Glu Thr Ser Phe TrpLeu 385 390 395 400 Leu Trp Ala Asn Ser Ala Val Asn Pro Val Leu Tyr ProLeu Cys His 405 410 415 Tyr Ser Phe Arg Arg Ala Phe Thr Lys Leu Leu CysPro Gln Lys Leu 420 425 430 Lys Val Gln Pro His Gly Ser Leu Glu Gln CysTrp Lys 435 440 445 1338 base pairs nucleic acid single linear cDNA CDS1..1335 6 ATG GAG CGC GCG CCG CCC GAC GGG CTG ATG AAC GCG TCG GGC ACTCTG 48 Met Glu Arg Ala Pro Pro Asp Gly Leu Met Asn Ala Ser Gly Thr Leu 15 10 15 GCC GGA GAG GCG GCG GCT GCA GGC GGG GCG CGC GGC TTC TCG GCT GCC96 Ala Gly Glu Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala 20 2530 TGG ACC GCT GTC CTG GCT GCG CTC ATG GCG CTG CTC ATC GTG GCC ACA 144Trp Thr Ala Val Leu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr 35 40 45GTA CTG GGC AAC GCG CTG GTC ATG CTC GCC TTC GTG GCG GAT TCG AGC 192 ValLeu Gly Asn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser 50 55 60 CTCCGC ACC CAG AAC AAC TTC TTT CTG CTC AAC CTC GCC ATC TCC GAC 240 Leu ArgThr Gln Asn Asn Phe Phe Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 TTCCTC GTG GGT GCC TTC TGC ATC CCA TTG TAC GTA CCC TAT GTG CTG 288 Phe LeuVal Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu 85 90 95 ACC GGCCGT TGG ACC TTC GGC CGG GGC CTC TGC AAG CTG TGG CTG GTG 336 Thr Gly ArgTrp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val 100 105 110 GTA GACTAC CTA CTG TGT GCC TCC TCG GTC TTC AAC ATC GTA CTC ATC 384 Val Asp TyrLeu Leu Cys Ala Ser Ser Val Phe Asn Ile Val Leu Ile 115 120 125 AGC TATGAC CGA TTC CTG TCA GTC ACT CGA GCT GTC TCC TAC AGG GCC 432 Ser Tyr AspArg Phe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala 130 135 140 CAG CAGGGG GAC ACG AGA CGG GCC GTT CGG AAG ATG GCA CTG GTG TGG 480 Gln Gln GlyAsp Thr Arg Arg Ala Val Arg Lys Met Ala Leu Val Trp 145 150 155 160 GTGCTG GCC TTC CTG CTG TAT GGG CCT GCC ATC CTG AGT TGG GAG TAC 528 Val LeuAla Phe Leu Leu Tyr Gly Pro Ala Ile Leu Ser Trp Glu Tyr 165 170 175 CTGTCT GGT GGC AGT TCC ATC CCC GAG GGC CAC TGC TAT GCT GAG TTC 576 Leu SerGly Gly Ser Ser Ile Pro Glu Gly His Cys Tyr Ala Glu Phe 180 185 190 TTCTAC AAC TGG TAC TTT CTC ATC ACG GCC TCC ACC CTC GAG TTC TTC 624 Phe TyrAsn Trp Tyr Phe Leu Ile Thr Ala Ser Thr Leu Glu Phe Phe 195 200 205 ACGCCC TTC CTC AGC GTT ACC TTC TTC AAC CTC AGC ATC TAC CTG AAC 672 Thr ProPhe Leu Ser Val Thr Phe Phe Asn Leu Ser Ile Tyr Leu Asn 210 215 220 ATCCAG AGG CGC ACC CGC CTT CGG CTT GAT GGG GGC CGT GAG GCT GGC 720 Ile GlnArg Arg Thr Arg Leu Arg Leu Asp Gly Gly Arg Glu Ala Gly 225 230 235 240CCA GAA CCC CCA CCA GAT GCC CAG CCC TCG CCA CCT CCA GCT CCC CCC 768 ProGlu Pro Pro Pro Asp Ala Gln Pro Ser Pro Pro Pro Ala Pro Pro 245 250 255AGC TGC TGG GGC TGC TGG CCA AAA GGG CAT GGC GAG GCC ATG CCG TTG 816 SerCys Trp Gly Cys Trp Pro Lys Gly His Gly Glu Ala Met Pro Leu 260 265 270CAC AGG TAT GGG GTG GGT GAG GCA GGC CCT GGT GTT GAG GCT GGG GAG 864 HisArg Tyr Gly Val Gly Glu Ala Gly Pro Gly Val Glu Ala Gly Glu 275 280 285GCT GCC CTC GGG GGT GGC AGT GGT GGA GGT GCT GCT GCC TCG CCC ACC 912 AlaAla Leu Gly Gly Gly Ser Gly Gly Gly Ala Ala Ala Ser Pro Thr 290 295 300TCC AGC TCT GGC AGC TCC TCA AGG GGC ACT GAG AGG CCA CGC TCA CTC 960 SerSer Ser Gly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu 305 310 315320 AAA AGG GGC TCC AAG CCA TCA GCA TCT TCA GCA TCC CTG GAG AAG CGC 1008Lys Arg Gly Ser Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg 325 330335 ATG AAG ATG GTG TCC CAG AGC ATC ACC CAG CGC TTC CGG CTG TCG CGG 1056Met Lys Met Val Ser Gln Ser Ile Thr Gln Arg Phe Arg Leu Ser Arg 340 345350 GAC AAG AAG GTG GCC AAG TCG CTG GCC ATC ATC GTG AGC ATC TTT GGG 1104Asp Lys Lys Val Ala Lys Ser Leu Ala Ile Ile Val Ser Ile Phe Gly 355 360365 CTC TGC TGG GCG CCG TAC ACG CTC CTA ATG ATC ATC CGA GCT GCT TGC 1152Leu Cys Trp Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys 370 375380 CAT GGC CGC TGC ATC CCC GAT TAC TGG TAC GAG ACG TCC TTC TGG CTT 1200His Gly Arg Cys Ile Pro Asp Tyr Trp Tyr Glu Thr Ser Phe Trp Leu 385 390395 400 CTG TGG GCC AAC TCG GCC GTC AAC CCC GTC CTC TAC CCA CTG TGC CAC1248 Leu Trp Ala Asn Ser Ala Val Asn Pro Val Leu Tyr Pro Leu Cys His 405410 415 TAC AGC TTC CGC AGA GCC TTC ACC AAG CTC CTC TGC CCC CAG AAG CTC1296 Tyr Ser Phe Arg Arg Ala Phe Thr Lys Leu Leu Cys Pro Gln Lys Leu 420425 430 AAG GTC CAG CCC CAC GGC TCC CTG GAG CAG TGC TGG AAG TGA 1338 LysVal Gln Pro His Gly Ser Leu Glu Gln Cys Trp Lys 435 440 445 26 aminoacids amino acid linear peptide internal 7 Thr Ala Val Leu Ala Ala LeuMet Ala Leu Leu Ile Val Ala Thr Val 1 5 10 15 Leu Gly Asn Ala Leu ValMet Leu Ala Phe 20 25 19 amino acids amino acid linear peptide internal8 Leu Leu Asn Leu Ala Ile Ser Asp Phe Leu Val Gly Ala Phe Cys Ile 1 5 1015 Pro Leu Tyr 22 amino acids amino acid linear peptide internal 9 LeuTrp Leu Val Val Asp Tyr Leu Leu Cys Thr Ser Ser Ala Phe Asn 1 5 10 15Ile Val Leu Ile Ser Tyr 20 23 amino acids amino acid linear peptideinternal 10 Ala Val Arg Lys Met Leu Leu Val Trp Val Leu Ala Phe Leu LeuTyr 1 5 10 15 Gly Pro Ala Ile Leu Ser Trp 20 23 amino acids amino acidlinear peptide internal 11 Tyr Phe Leu Ile Thr Ala Ser Thr Leu Glu PhePhe Thr Pro Phe Leu 1 5 10 15 Ser Val Thr Phe Phe Asn Leu 20 21 aminoacids amino acid linear peptide internal 12 Leu Ala Val Ile Val Ser IlePhe Gly Leu Cys Trp Ala Pro Tyr Thr 1 5 10 15 Leu Leu Met Ile Ile 20 21amino acids amino acid linear peptide internal 13 Thr Ser Phe Trp LeuLeu Trp Ala Asn Ser Ala Val Asn Pro Val Leu 1 5 10 15 Tyr Pro Leu CysHis 20 26 amino acids amino acid linear peptide internal 14 Thr Ala ValLeu Ala Ala Leu Met Ala Leu Leu Ile Val Ala Thr Val 1 5 10 15 Leu GlyAsn Ala Leu Val Met Leu Ala Phe 20 25 19 amino acids amino acid linearpeptide internal 15 Leu Leu Asn Leu Ala Ile Ser Asp Phe Leu Val Gly AlaPhe Cys Ile 1 5 10 15 Pro Leu Tyr 22 amino acids amino acid linearpeptide internal 16 Leu Trp Leu Val Val Asp Tyr Leu Leu Cys Ala Ser SerVal Phe Asn 1 5 10 15 Ile Val Leu Ile Ser Tyr 20 23 amino acids aminoacid linear peptide internal 17 Ala Val Arg Lys Met Ala Leu Val Trp ValLeu Ala Phe Leu Leu Tyr 1 5 10 15 Gly Pro Ala Ile Leu Ser Trp 20 23amino acids amino acid linear peptide internal 18 Tyr Phe Leu Ile ThrAla Ser Thr Leu Glu Phe Phe Thr Pro Phe Leu 1 5 10 15 Ser Val Thr PhePhe Asn Leu 20 21 amino acids amino acid linear peptide internal 19 LeuAla Ile Ile Val Ser Ile Phe Gly Leu Cys Trp Ala Pro Tyr Thr 1 5 10 15Leu Leu Met Ile Ile 20 21 amino acids amino acid linear peptide internal20 Thr Ser Phe Trp Leu Leu Trp Ala Asn Ser Ala Val Asn Pro Val Leu 1 510 15 Tyr Pro Leu Cys His 20 17 base pairs nucleic acid single linearcDNA 21 CCTGCGGGGC CATGGAG 17 17 base pairs nucleic acid single linearcDNA 22 GTGGCCCACC AGAGCCT 17 17 base pairs nucleic acid single linearcDNA 23 CAGCCACGCC TCTCTCA 17 18 base pairs nucleic acid single linearcDNA 24 GCCTGCTGGG CCATGGAG 18 16 base pairs nucleic acid single linearcDNA 25 TGAGCAGCTG CCCCAC 16 16 base pairs nucleic acid single linearcDNA 26 CTGAGGCCAG GCCCTT 16 20 base pairs nucleic acid single linearcDNA 27 CAAGAACCCT TTAAGCCAAG 20 20 base pairs nucleic acid singlelinear cDNA 28 GAAGAAGGTA ACGCTGAGGA 20 20 base pairs nucleic acidsingle linear cDNA 29 CAGAACCCCC ACCAGATGCC 20 20 base pairs nucleicacid single linear cDNA 30 TAGTGGCACA GTGGGTAGAG 20 2218 base pairsnucleic acid single linear cDNA CDS 119..1204 31 GGT GCC TTC TGC ATC CCATTG TAC GTA CCC TAT GTG CTG ACC GGC CGT 48 Gly Ala Phe Cys Ile Pro LeuTyr Val Pro Tyr Val Leu Thr Gly Arg 1 5 10 15 TGG ACC TTC GGC CGG GGCCTC TGC AAG CTG TGG CTG GTG GTA GAC TAC 96 Trp Thr Phe Gly Arg Gly LeuCys Lys Leu Trp Leu Val Val Asp Tyr 20 25 30 CTA CTG TGT GCC TCC TCG GTCTTC AAC ATC GTA CTC ATC AGC TAT GAC 144 Leu Leu Cys Ala Ser Ser Val PheAsn Ile Val Leu Ile Ser Tyr Asp 35 40 45 CGA TTC CTG TCA GTC ACT CGA GCTGTC TCC TAC AGG GCC CAG CAG GGG 192 Arg Phe Leu Ser Val Thr Arg Ala ValSer Tyr Arg Ala Gln Gln Gly 50 55 60 GAC ACG AGA CGG GCC GTT CGG AAG ATGGCA CTG GTG TGG GTG CTG GCC 240 Asp Thr Arg Arg Ala Val Arg Lys Met AlaLeu Val Trp Val Leu Ala 65 70 75 80 TTC CTG CTG TAT GGG CCT GCC ATC CTGAGT TGG GAG TAC CTG TCT GGT 288 Phe Leu Leu Tyr Gly Pro Ala Ile Leu SerTrp Glu Tyr Leu Ser Gly 85 90 95 GGC AGT TCC ATC CCC GAG GGC CAC TGC TATGCT GAG TTC TTC TAC AAC 336 Gly Ser Ser Ile Pro Glu Gly His Cys Tyr AlaGlu Phe Phe Tyr Asn 100 105 110 TGG TAC TTT CTC ATC TCG GCC TCC ACC CTCGAG TTC TTC ACG CCC TTC 384 Trp Tyr Phe Leu Ile Ser Ala Ser Thr Leu GluPhe Phe Thr Pro Phe 115 120 125 CTC AGC GTT ACC TTC TTC AAC CTC AGC ATCTAC CTG AAC ATC CAG AGG 432 Leu Ser Val Thr Phe Phe Asn Leu Ser Ile TyrLeu Asn Ile Gln Arg 130 135 140 CGC ACC CGC CTT CGG CTT GAT GGG GGC CGTGAG GCT GGC CCA GAA CCC 480 Arg Thr Arg Leu Arg Leu Asp Gly Gly Arg GluAla Gly Pro Glu Pro 145 150 155 160 CCA CCA GAT GCC CAG CCC TCG CCA CCTCCA GCT CCC CCC AGC TGC TGG 528 Pro Pro Asp Ala Gln Pro Ser Pro Pro ProAla Pro Pro Ser Cys Trp 165 170 175 GGC TGC TGG CCA AAA GGG CAT GGC GAGGCC ATG CCG TTG CAC AGG TAT 576 Gly Cys Trp Pro Lys Gly His Gly Glu AlaMet Pro Leu His Arg Tyr 180 185 190 GGG GTG GGT GAG GCA GGC CCT GGT GTTGAG GCT GGG GAG GCT GCC CTC 624 Gly Val Gly Glu Ala Gly Pro Gly Val GluAla Gly Glu Ala Ala Leu 195 200 205 GGG GGT GGC AGT GGT GGA GGT GCT GCTGCC TCG CCC ACC TCC AGC TCT 672 Gly Gly Gly Ser Gly Gly Gly Ala Ala AlaSer Pro Thr Ser Ser Ser 210 215 220 GGC AGC TCC TCA AGG GGC ACT GAG AGGCCA CGC TCA CTC AAA AGG GGC 720 Gly Ser Ser Ser Arg Gly Thr Glu Arg ProArg Ser Leu Lys Arg Gly 225 230 235 240 TCC AAG CCA TCA GCA TCT TCA GCATCC CTG GAG AAG CGC ATG AAG ATG 768 Ser Lys Pro Ser Ala Ser Ser Ala SerLeu Glu Lys Arg Met Lys Met 245 250 255 GTG TCC CAG AGC ATC ACC CAG CGCTTC CGG CTG TCG CGG GAC AAG AAG 816 Val Ser Gln Ser Ile Thr Gln Arg PheArg Leu Ser Arg Asp Lys Lys 260 265 270 GTG GCC AAG TCG CTG GCC ATC ATCGTG AGC ATC TTT GGG CTC TGC TGG 864 Val Ala Lys Ser Leu Ala Ile Ile ValSer Ile Phe Gly Leu Cys Trp 275 280 285 GCG CCG TAC ACG CTC CTA ATG ATCATC CGA GCT GCT TGC CAT GGC CGC 912 Ala Pro Tyr Thr Leu Leu Met Ile IleArg Ala Ala Cys His Gly Arg 290 295 300 TGC ATC CCC GAT TAC TGG TAC GAGACG TCC TTC TGG CTT CTG TGG GCC 960 Cys Ile Pro Asp Tyr Trp Tyr Glu ThrSer Phe Trp Leu Leu Trp Ala 305 310 315 320 AAC TCG GCC GTC AAC CCC GTCCTC TAC CCA CTG TGC CAC TAC AGC TTC 1008 Asn Ser Ala Val Asn Pro Val LeuTyr Pro Leu Cys His Tyr Ser Phe 325 330 335 CGC AGA GCC TTC ACC AAG CTCCTC TGC CCC CAG AAG CTC AAG GTC CAG 1056 Arg Arg Ala Phe Thr Lys Leu LeuCys Pro Gln Lys Leu Lys Val Gln 340 345 350 CCC CAC GGC TCC CTG GAG CAGTGC TGG AAG TGAGCAGCTG CCCCACCCTT 1106 Pro His Gly Ser Leu Glu Gln CysTrp Lys 355 360 CTGAGGCCAG GCCCTTGTAC TTGTTTGAGT GGGCAGCCGG AGCGTGGGCGGGGCCCTGGT 1166 CCATGCTCCG CTCCAAATGC CATGGCGGCC TCTTAGATCA TCAACCCCGCAGTGGGGTAG 1226 CATGGCAGGT GGGCCAAGAG CCCTAGTTGG TGGAGCTAGA GTGTGCTGGTTAGCTCTGCC 1286 GCCACATTCT CCTTCACCAC ACAGAAGAGA CAATCCAGGA GTCCCAGGCATGCCTTCCAC 1346 CTACACACAC ACACACACAC ACACACACAC ACACACCACA GTGCAGTGCCAGTGATGTCC 1406 CCTTTTGCAT ATTTAGTGGT TGGTGTCCTC CCTAATGCAA ACCTCGGTGTGTGCTCCCGG 1466 CTCCGGCCCT GGCAATGCGT GCGTGCGCCC TGCATGTGCT CACACCCGCCACACACCCGC 1526 CCGCCACACA CTTGCAACAC CTCCTCTCTC CCAGAAGAGC TGGGGACGATGCCCTTTGCT 1586 GCCACTGTCT CTTGCTTAAT CCCAGAGCCT GGCTCCTTAT CCCCCACTCTCCCTTCAACT 1646 CTGCCCCACA AAGTGTCGAG CGCCTCGGGA AACTTGAAGC TTCTCTGCTCCTTCCACTCT 1706 GGATGTTTTC AGGAAGATGG AGGAGAAGAA AACACGTCTG TGAACTTGATGTTCCTTGGA 1766 TGTTTAATCA AGAGAGACAA AATTGCCGAG GAGCTCGGGG CTGGATTGGCAGGTGTGGGC 1826 TCCCACGCCC TCCTCCCTCA GTGCTGCAGC TTCCGGCTGA GCCGCGCCAGCTGCTTCTGC 1886 CTGCCCCGCC CCCAGGCTTG GGACGATGGC CCTGCCCTGC TTGCCCCGTCTGTACAATCA 1946 GAATTTGGGG GTGGGTGGTT ATGGGGTAGA GCGGCTCTTC ACTGTGCCCTAAAGGTCCTG 2006 AGGCTCACAG GACAGTCAGC AGGAGAGCAG GCAGGCCCGC GACACCTGGGAGGAATGCTT 2066 TGCCTCGTCC TGTGTACTCA CCTCAGGCTT CTGCATGCTC TGCTGCCCTTGTGCCCTGGT 2126 GTGCTGCCTC TGCCAATGTG AAAACACAAT AAAGTGTATT TTTTTAAAAAAAAAAAAAAA 2186 AAAAAAAAAA AAAAAAAAAA AAGGGCGGCC GC 2218 362 amino acidsamino acid linear protein 32 Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro TyrVal Leu Thr Gly Arg 1 5 10 15 Trp Thr Phe Gly Arg Gly Leu Cys Lys LeuTrp Leu Val Val Asp Tyr 20 25 30 Leu Leu Cys Ala Ser Ser Val Phe Asn IleVal Leu Ile Ser Tyr Asp 35 40 45 Arg Phe Leu Ser Val Thr Arg Ala Val SerTyr Arg Ala Gln Gln Gly 50 55 60 Asp Thr Arg Arg Ala Val Arg Lys Met AlaLeu Val Trp Val Leu Ala 65 70 75 80 Phe Leu Leu Tyr Gly Pro Ala Ile LeuSer Trp Glu Tyr Leu Ser Gly 85 90 95 Gly Ser Ser Ile Pro Glu Gly His CysTyr Ala Glu Phe Phe Tyr Asn 100 105 110 Trp Tyr Phe Leu Ile Ser Ala SerThr Leu Glu Phe Phe Thr Pro Phe 115 120 125 Leu Ser Val Thr Phe Phe AsnLeu Ser Ile Tyr Leu Asn Ile Gln Arg 130 135 140 Arg Thr Arg Leu Arg LeuAsp Gly Gly Arg Glu Ala Gly Pro Glu Pro 145 150 155 160 Pro Pro Asp AlaGln Pro Ser Pro Pro Pro Ala Pro Pro Ser Cys Trp 165 170 175 Gly Cys TrpPro Lys Gly His Gly Glu Ala Met Pro Leu His Arg Tyr 180 185 190 Gly ValGly Glu Ala Gly Pro Gly Val Glu Ala Gly Glu Ala Ala Leu 195 200 205 GlyGly Gly Ser Gly Gly Gly Ala Ala Ala Ser Pro Thr Ser Ser Ser 210 215 220Gly Ser Ser Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu Lys Arg Gly 225 230235 240 Ser Lys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg Met Lys Met245 250 255 Val Ser Gln Ser Ile Thr Gln Arg Phe Arg Leu Ser Arg Asp LysLys 260 265 270 Val Ala Lys Ser Leu Ala Ile Ile Val Ser Ile Phe Gly LeuCys Trp 275 280 285 Ala Pro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala CysHis Gly Arg 290 295 300 Cys Ile Pro Asp Tyr Trp Tyr Glu Thr Ser Phe TrpLeu Leu Trp Ala 305 310 315 320 Asn Ser Ala Val Asn Pro Val Leu Tyr ProLeu Cys His Tyr Ser Phe 325 330 335 Arg Arg Ala Phe Thr Lys Leu Leu CysPro Gln Lys Leu Lys Val Gln 340 345 350 Pro His Gly Ser Leu Glu Gln CysTrp Lys 355 360 1086 base pairs nucleic acid single linear cDNA CDS1..1086 33 GGT GCC TTC TGC ATC CCA TTG TAC GTA CCC TAT GTG CTG ACC GGCCGT 48 Gly Ala Phe Cys Ile Pro Leu Tyr Val Pro Tyr Val Leu Thr Gly Arg 15 10 15 TGG ACC TTC GGC CGG GGC CTC TGC AAG CTG TGG CTG GTG GTA GAC TAC96 Trp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val Val Asp Tyr 20 2530 CTA CTG TGT GCC TCC TCG GTC TTC AAC ATC GTA CTC ATC AGC TAT GAC 144Leu Leu Cys Ala Ser Ser Val Phe Asn Ile Val Leu Ile Ser Tyr Asp 35 40 45CGA TTC CTG TCA GTC ACT CGA GCT GTC TCC TAC AGG GCC CAG CAG GGG 192 ArgPhe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala Gln Gln Gly 50 55 60 GACACG AGA CGG GCC GTT CGG AAG ATG GCA CTG GTG TGG GTG CTG GCC 240 Asp ThrArg Arg Ala Val Arg Lys Met Ala Leu Val Trp Val Leu Ala 65 70 75 80 TTCCTG CTG TAT GGG CCT GCC ATC CTG AGT TGG GAG TAC CTG TCT GGT 288 Phe LeuLeu Tyr Gly Pro Ala Ile Leu Ser Trp Glu Tyr Leu Ser Gly 85 90 95 GGC AGTTCC ATC CCC GAG GGC CAC TGC TAT GCT GAG TTC TTC TAC AAC 336 Gly Ser SerIle Pro Glu Gly His Cys Tyr Ala Glu Phe Phe Tyr Asn 100 105 110 TGG TACTTT CTC ATC TCG GCC TCC ACC CTC GAG TTC TTC ACG CCC TTC 384 Trp Tyr PheLeu Ile Ser Ala Ser Thr Leu Glu Phe Phe Thr Pro Phe 115 120 125 CTC AGCGTT ACC TTC TTC AAC CTC AGC ATC TAC CTG AAC ATC CAG AGG 432 Leu Ser ValThr Phe Phe Asn Leu Ser Ile Tyr Leu Asn Ile Gln Arg 130 135 140 CGC ACCCGC CTT CGG CTT GAT GGG GGC CGT GAG GCT GGC CCA GAA CCC 480 Arg Thr ArgLeu Arg Leu Asp Gly Gly Arg Glu Ala Gly Pro Glu Pro 145 150 155 160 CCACCA GAT GCC CAG CCC TCG CCA CCT CCA GCT CCC CCC AGC TGC TGG 528 Pro ProAsp Ala Gln Pro Ser Pro Pro Pro Ala Pro Pro Ser Cys Trp 165 170 175 GGCTGC TGG CCA AAA GGG CAT GGC GAG GCC ATG CCG TTG CAC AGG TAT 576 Gly CysTrp Pro Lys Gly His Gly Glu Ala Met Pro Leu His Arg Tyr 180 185 190 GGGGTG GGT GAG GCA GGC CCT GGT GTT GAG GCT GGG GAG GCT GCC CTC 624 Gly ValGly Glu Ala Gly Pro Gly Val Glu Ala Gly Glu Ala Ala Leu 195 200 205 GGGGGT GGC AGT GGT GGA GGT GCT GCT GCC TCG CCC ACC TCC AGC TCT 672 Gly GlyGly Ser Gly Gly Gly Ala Ala Ala Ser Pro Thr Ser Ser Ser 210 215 220 GGCAGC TCC TCA AGG GGC ACT GAG AGG CCA CGC TCA CTC AAA AGG GGC 720 Gly SerSer Ser Arg Gly Thr Glu Arg Pro Arg Ser Leu Lys Arg Gly 225 230 235 240TCC AAG CCA TCA GCA TCT TCA GCA TCC CTG GAG AAG CGC ATG AAG ATG 768 SerLys Pro Ser Ala Ser Ser Ala Ser Leu Glu Lys Arg Met Lys Met 245 250 255GTG TCC CAG AGC ATC ACC CAG CGC TTC CGG CTG TCG CGG GAC AAG AAG 816 ValSer Gln Ser Ile Thr Gln Arg Phe Arg Leu Ser Arg Asp Lys Lys 260 265 270GTG GCC AAG TCG CTG GCC ATC ATC GTG AGC ATC TTT GGG CTC TGC TGG 864 ValAla Lys Ser Leu Ala Ile Ile Val Ser Ile Phe Gly Leu Cys Trp 275 280 285GCG CCG TAC ACG CTC CTA ATG ATC ATC CGA GCT GCT TGC CAT GGC CGC 912 AlaPro Tyr Thr Leu Leu Met Ile Ile Arg Ala Ala Cys His Gly Arg 290 295 300TGC ATC CCC GAT TAC TGG TAC GAG ACG TCC TTC TGG CTT CTG TGG GCC 960 CysIle Pro Asp Tyr Trp Tyr Glu Thr Ser Phe Trp Leu Leu Trp Ala 305 310 315320 AAC TCG GCC GTC AAC CCC GTC CTC TAC CCA CTG TGC CAC TAC AGC TTC 1008Asn Ser Ala Val Asn Pro Val Leu Tyr Pro Leu Cys His Tyr Ser Phe 325 330335 CGC AGA GCC TTC ACC AAG CTC CTC TGC CCC CAG AAG CTC AAG GTC CAG 1056Arg Arg Ala Phe Thr Lys Leu Leu Cys Pro Gln Lys Leu Lys Val Gln 340 345350 CCC CAC GGC TCC CTG GAG CAG TGC TGG AAG 1086 Pro His Gly Ser Leu GluGln Cys Trp Lys 355 360 8 amino acids amino acid linear protein 34 GlyAla Phe Cys Ile Pro Leu Tyr 1 5 22 amino acids amino acid linear protein35 Leu Trp Leu Val Val Asp Tyr Leu Leu Cys Ala Ser Ser Val Phe Asn 1 510 15 Ile Val Leu Ile Ser Tyr 20 23 amino acids amino acid linearprotein 36 Ala Val Arg Lys Met Ala Leu Val Trp Val Leu Ala Phe Leu LeuTyr 1 5 10 15 Gly Pro Ala Ile Leu Ser Trp 20 23 amino acids amino acidlinear protein 37 Tyr Phe Leu Ile Ser Ala Ser Thr Leu Glu Phe Phe ThrPro Phe Leu 1 5 10 15 Ser Val Thr Phe Phe Asn Leu 20 21 amino acidsamino acid linear protein 38 Leu Ala Ile Ile Val Ser Ile Phe Gly Leu CysTrp Ala Pro Tyr Thr 1 5 10 15 Leu Leu Met Ile Ile 20 21 amino acidsamino acid linear protein 39 Thr Ser Phe Trp Leu Leu Trp Ala Asn Ser AlaVal Asn Pro Val Leu 1 5 10 15 Tyr Pro Leu Cys His 20

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2; b) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:5; c) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:32; d) anucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:2; e) anucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:5; f) anucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:32, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:32; g) anucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, wherein the nucleic acid molecule hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1 under stringent conditions; and h) anucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the nucleic acid molecule hybridizes to a nucleicacid-molecule comprising SEQ ID NO:4 under stringent conditions; and i)a nucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:32, wherein the nucleic acid molecule hybridizes to a nucleic acidmolecule comprising SEQ ID NO:31 under stringent conditions.
 2. Thenucleic acid molecule of claim 1 further comprising vector nucleic acidsequences.
 3. The nucleic acid molecule of claim 1 further comprisingnucleic acid sequences encoding a heterologous polypeptide.
 4. A hostcell which contains the nucleic acid molecule of claim
 1. 5. The hostcell of claim 4 which is a mammalian host cell.
 6. A non-human mammalianhost cell containing the nucleic acid molecule of claim
 1. 7. Theisolated nucleic acid molecule of claim 1, which is selected from thegroup consisting of: a) the coding region of SEQ ID NO:1; b) the codingregion of SEQ ID NO:4; c) the coding region of SEQ ID NO:31; d) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:7; e) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:8; f) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:9; g) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:10; h) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:11; i) a nucleic acid molecule which encodesa polypeptide comprising the amino acid sequence of SEQ ID NO:12; and j)a nucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:13.
 8. An isolated polypeptide selected fromthe group consisting of: a) a polypeptide comprising the amino acidsequence of SEQ ID NO:2; b) a polypeptide comprising the amino acidsequence of SEQ ID NO:5; c) a polypeptide comprising the amino acidsequence of SEQ ID NO:32; d) a fragment of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the fragment comprises atleast 15 contiguous amino acids of SEQ ID NO:2; e) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:5, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:5; f) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:32, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:32; g) a naturally occurring allelic variant ofa polypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:1 under stringentconditions; h) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO:4 under stringent conditions;and i) a naturally occurring allelic variant of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:31 under stringent conditions.
 9. Thepolypeptide of claim 8, further comprising heterologous amino acidsequences.
 10. An antibody which selectively binds to a polypeptide ofclaim
 8. 11. A method for producing a polypeptide selected from thegroup consisting of: a) a polypeptide comprising the amino acid sequenceof SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence ofSEQ ID NO:5; c) a polypeptide comprising the amino acid sequence of SEQID NO:32; d) a fragment of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the fragment comprises at least 15contiguous amino acids of SEQ ID NO:2; e) a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:5; f) afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:32, wherein the fragment comprises at least 15 contiguous amino acidsof SEQ ID NO:32; g) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:1 under stringentconditions; h) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO:4 under stringent conditions;and i) a naturally occurring allelic variant of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:31 under stringent conditions; the methodcomprising the step of culturing the host cell of claim 4 underconditions in which the nucleic acid molecule is expressed.
 12. A methodfor detecting the presence of a polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:2; b) a polypeptide comprising the amino acid sequence of SEQID NO:5; c) a polypeptide comprising the amino acid sequence of SEQ IDNO:32; d) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:2, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:2; e) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:5, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:5; f) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:32; g) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO:1 under stringent conditions;h) a naturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:5, wherein the polypeptide is encodedby a nucleic acid molecule which hybridizes to a nucleic acid moleculecomprising SEQ ID NO:4 under stringent conditions; and i) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:32, wherein the polypeptide is encoded by anucleic acid molecule which hybridizes to a nucleic acid moleculecomprising SEQ ID NO:31 under stringent conditions; in a sample, themethod comprising the steps of: i) contacting the sample with a compoundwhich selectively binds to the polypeptide; and ii) determining whetherthe compound binds to the polypeptide in the sample.
 13. The method ofclaim 12, wherein the compound which binds to the polypeptide is anantibody.
 14. A kit comprising reagents used for the method of claim 12,wherein the reagents comprise a compound which selectively binds to apolypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; b) a polypeptidecomprising the amino acid sequence of SEQ ID NO:5; c) a polypeptidecomprising the amino acid sequence of SEQ ID NO:32; d) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:2; e) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:5; f) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:32; g) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:1 under stringent conditions; h) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 understringent conditions; and i) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:31 under stringentconditions.
 15. A method for detecting the presence of a nucleic acidmolecule selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; b) a polypeptidecomprising the amino acid sequence of SEQ ID NO:5; c) a polypeptidecomprising the amino acid sequence of SEQ ID NO:32; d) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:2; e) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:5; f) a fragment of a polypeptide comprisingthe amino-acid sequence of SEQ ID NO:32, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:32; g) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:1 under stringent conditions; h) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 understringent conditions; and i) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:31 under stringentconditions; in a sample, the method comprising the steps of: i)contacting the sample with a nucleic acid probe or primer whichselectively hybridizes to the nucleic acid molecule; and ii) determiningwhether the nucleic acid probe or primer binds to a nucleic acidmolecule in the sample.
 16. The method of claim 15, wherein the samplecomprises mRNA molecules and is contacted with a nucleic acid probe. 17.A kit comprising reagents used for the method of claim 15, wherein thereagents comprise a compound which selectively hybridizes to a nucleicacid molecule selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; b) a polypeptidecomprising the amino acid sequence of SEQ ID NO:5; c) a polypeptidecomprising the amino acid sequence of SEQ ID NO:32; d) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:2; e) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:5; f) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:32; g) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:1 under stringent conditions; h) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 understringent conditions; and i) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:31 under stringentconditions.
 18. A method for identifying a compound which binds to apolypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; b) a polypeptidecomprising the amino acid sequence of SEQ ID NO:5; c) a polypeptidecomprising the amino acid sequence of SEQ ID NO:32; d) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:2; e) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:5; f) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:32; g) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:1 under stringent conditions; h) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 understringent conditions; and i) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:31 under stringentconditions, the method comprising the steps of: i) contacting thepolypeptide, or a cell expressing the polypeptide with a test compound;and ii) determining whether the polypeptide binds to the test compound.19. The method of claim 18, wherein the binding of the test compound tothe polypeptide is detected by a method selected from the groupconsisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay formACHR-6 activity.
 20. A method for modulating the activity of apolypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; b) a polypeptidecomprising the amino acid sequence of SEQ ID NO:5; c) a polypeptidecomprising the amino acid sequence of SEQ ID NO:32; d) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:2; e) a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:5; f) a fragment of a polypeptide comprisingthe amino acid sequence of SEQ ID NO:32, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:32; g) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:1 under stringent conditions; h) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:5, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 understringent conditions; and i) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:32, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:31 under stringentconditions, the method comprising the steps of: i) contacting a cellexpressing the polypeptide with a compound which binds to thepolypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.
 21. The method of claim 20, wherein the activity is aphosphatidylinositol activity.
 22. The method of claim 20, wherein themethod results in an increase in phosphatidylinositol metabolism. 23.The method of claim 20, wherein the method results in a decrease inphosphatidylinositol metabolism.